Crosslinked compounds and methods of making and using thereof

ABSTRACT

Described herein are crosslinked compounds useful in numerous treatments. Described herein are methods of making crosslinked compounds via (1) the oxidative coupling of two or more thiol compounds or (2) by the reaction between at least one tbiol compound with at least one thiol-reactive compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/390,504, filed Jun. 21, 2002. This application is herebyincorporated by this reference in its entirety for all of its teachings.

The research leading to this invention was funded in part by theNational Institutes of Health, Grant No. NIH 5R01 DC04663. The U.S.Government may have certain rights in this invention.

BACKGROUND

The use of macromolecules in pharmaceutical applications has receivedconsiderable attention. At times, it is desirable to couple two or moremacromolecules to produce new macromolecule scaffolds with multipleactivities. Existing technologies used to couple two or macromolecules,however, present numerous difficulties. For example, the alkalineconditions or high temperatures necessary to create hydrogels with highmechanical strength are cumbersome and harsh. Although the use ofcrosslinkers to produce macromolecular scaffolds has met with somesuccess, the crosslinking agents are often relatively small, cytotoxicmolecules, and the resulting scaffold has to be extracted or washedextensively to remove traces of unreacted reagents and byproducts(Hennink, W. E.; van Nostrum, C. F. Adv. Drug Del. Rev. 2002, 54,13-36), thus precluding use in many medical applications. Aphysiologically compatible macromolecular scaffold capable of beingproduced in a straightforward manner is needed before they will beuseful as therapeutic aids. Described herein are compounds and methodsthat are capable of coupling two or more molecules, such asmacromolecules, under mild conditions.

SUMMARY OF EMBODIMENTS

Described herein are crosslinked compounds. Also described herein aremethods of making and using crosslinked compounds.

The advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 shows the reaction scheme for producing HA-thiolated derivatives.

FIG. 2 shows (a) absorption at 242 nm as a function of pH for HA-DTPHand HA-DTBH solution and (b) logarithmic plot oflog[(A_(max)−A_(i))/A_(i)] vs. pH. The pK_(a) values correspond to theintercept with the abscissa.

FIG. 3 shows the swelling of HA-DTPH and HA-DTBH films in PBS at pH 7.4.The open circles and triangles are the films coupled via oxidation with0.3% H₂O₂ after air oxidation, and the closed circles and triangles arefilms coupled by air oxidation only.

FIG. 4 shows the disulfide content in HA-DTPH and HA-DTBH films. Key:a=air oxidation only; b=air oxidation followed by oxidation with H₂O₂.

FIG. 5 shows the release of blue dextran from HA-DTPH in PBS containingdifferent concentrations of DTT at pH 7.4.

FIG. 6 shows fibroblast proliferation in HA-DTPH hydrogel after in vitroculture of 0, 1, 2, and 3 days.

FIG. 7 shows the synthesis of thiolated HA and gelatin.

FIG. 8 shows the effect of salt concentration on polyelectrolyte complexformation in mixed HA-gelatin solutions. HA-DTPH and gelatin-DTPH, both3.0% (w/v), were dissolved in 0.02 M PBS, the pH was adjusted to 7.4(3.0% w/v), and the solutions were then mixed at different ratios. Theabsorption was determined at 15 min (open circles, no added salt) and 2h (open diamonds, 1.0% NaCl) after the preparation of solution in 0.5-cmspectrophotometer cell.

FIG. 9 shows the determination of disulfide density in HA-DTPHgelatin-DTPH hydrogel films. The hydrogel films were prepared with 3.0%(w/v) polymer in 0.02 M PBS (pH 7.4) with 1.0% (w/v) NaCl and thenexhaustively hydrolyzed in acid (n=3). NTSB and DTNB reagents were usedas described to obtain total sulfur and thiol contents. The theoreticaldisulfide density (open circles) was calculated from thiol density(HA-DTPH 0.77 mmol/g, gelatin-DTPH 0.51 mmol/g).

FIG. 10 shows the equilibrium swelling ratio of HA-gelatin films. Theratio was measured in PBS at 37° C., 300 rpm (n=3). The hydrogel filmswere prepared with 3.0% (w/v) polymer in 0.02 M PBS (pH 7.4) with 1.0%(w/v) NaCl.

FIG. 11 shows the enzymatic degradation of mixed HA-gelatin films. Theweight loss of HA-gelatin hydrogel films in 300 U/ml enzyme solutions(HAse, open triangles; collagenase, open squares; open circles, HAseplus collagenase) at 37° C., 150 rpm (n=3). Panel A: HA-gelatin, 20:80.Panel B: HA-gelatin, 40:60. The hydrogel films were prepared with 3.0%(w/v) polymer in 0.02 M PBS (pH 7.4) with 1.0% (w/v) NaCl.

FIG. 12 shows the cell attachment and spreading of fibroblasts ofHA-gelatin films. Fluorescent microscopic images of adherent and spreadBalb/c 3T3 fibroblast on the surface of HA-gelatin hydrogel films after24 h of in vitro culture. The cells were initially seeded at 25,000cells/cm2 and were stained with F-DA. Panel a: 100% HA film; Panel b:HA-gelatin, 80:20; Panel c: HA-gelatin, 40:60 film; and Panel d: 100%gelatin-DTPH film. Original magnification: Panels a, b, c, and d at×100.

FIG. 13 shows the proliferation of Balb/c 3T3 fibroblast on the surfaceof HA-gelatin hydrogel film. The cells were initially seeded at 5,000cells/cm² and the cell number was determined by MTT assay after one dayand three days culture in vitro (n=5). Tissue culture polystyrene (PS)was used as control, and the relative cell density on tissue culturepolystyrene after one day of in vitro culture was defined as 1.0. Theinset shows the proliferation ratio (PR) as a function of percentgelatin (% G) in the hydrogel.

FIG. 14 shows structures of α, β-unsaturated esters and amides ofpoly(ethylene glycol) crosslinked with thiolated HA and thiolatedgelatin.

FIG. 15 shows the conjugate addition between PEGDA, PEGDM, PEGDAA,PEGDMA and cysteine.

FIG. 16 shows the conjugate addition of HA-DTPH, HA-DTBH andPEG-acrylate.

FIG. 17 shows the digestion of HA-DTPH-PEGDA with HAse.

FIG. 18 shows the viability of T31 fibroblasts after 28 days in vitroculture in HA-DTPH-PEGDA hydrogel, confocal microscope,magnification=×200.

FIG. 19 shows the proliferation of T31 fibroblasts in HA-DTPH-PEGDA gel.

FIG. 20 shows the gross view of explants of HA-DTPH-PEGDA seeded with T31 fibroblasts after subcutaneous implantation in vivo in nude mice.

FIG. 21 shows the histological examination of the explants afterincubation in nude mice for 2 weeks (Panel A), 4 weeks (Panel B), and 8weeks (Panel C), immunohistochemistry (fibronectin). Originalmagnification×200.

FIG. 22 shows the synthesis of HA-DTPH-MMC.

FIG. 23 shows the synthesis of HA-DTPH-PEGDA-MMC.

FIGS. 24 a and 24 b show the results of in vitro MMC release results.

DETAILED DESCRIPTION

Before the present compounds, compositions, and/or methods are disclosedand described, it is to be understood that the aspects described beloware not limited to specific compounds, synthetic methods, or uses assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally substituted lower alkyl”means that the lower alkyl group can or can not be substituted and thatthe description includes both unsubstituted lower alkyl and lower alkylwhere there is substitution.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

References in the specification and concluding claims to parts byweight, of a particular element or component in a composition orarticle, denotes the weight relationship between the element orcomponent and any other elements or components in the composition orarticle for which a part by weight is expressed. Thus, in a compoundcontaining 2 parts by weight of component X and 5 parts by weightcomponent Y, X and Y are present at a weight ratio of 2:5, and arepresent in such ratio regardless of whether additional components arecontained in the compound.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. For example, apolysaccharide that contains at least one —COOH group can be representedby the formula Y—COOH, where Y is the remainder (i.e., residue) of thepolysaccharide molecule.

Variables such as R³-R⁵, R⁷, R⁸, E, L, J, G, M, Q, U, V, X, Y, and Zused throughout the application are the same variables as previouslydefined unless stated to the contrary.

The term “alkyl group” as used herein is a branched or unbranchedsaturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and thelike. A “lower alkyl” group is an alkyl group containing from one to sixcarbon atoms.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula —(CH₂)_(n)—, where n is an integer of from 2to 25.

The term “polyether group” as used herein is a group having the formula—[(CHR)_(n)O]_(m)—, where R is hydrogen or a lower alkyl group, n is aninteger of from 1 to 20, and m is an integer of from 1 to 100. Examplesof polyether groups include, polyethylene oxide, polypropylene oxide,and polybutylene oxide.

The term “polythioether group” as used herein is a group having theformula —[(CHR)_(n)S]_(m)—, where R is hydrogen or a lower alkyl group,n is an integer of from 1 to 20, and m is an integer of from 1 to 100.

The term “polyimino group” as used herein is a group having the formula—[(CHR)_(n)NR]_(m)—, where each R is, independently, hydrogen or a loweralkyl group, n is an integer of from 1 to 20, and m is an integer offrom 1 to 100.

The term “polyester group” as used herein is a group that is produced bythe reaction between a compound having at least two carboxylic acidgroups with a compound having at least two hydroxyl groups.

The term “polyamide group” as used herein is a group that is produced bythe reaction between a compound having at least two carboxylic acidgroups with a compound having at least two unsubstituted ormonosubstituted amino groups.

The term “aryl group” as used herein is any carbon-based aromatic groupincluding, but not limited to, benzene, naphthalene, etc. The term“aromatic” also includes “heteroaryl group,” which is defined as anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. The aryl group canbe substituted or unsubstituted. The aryl group can be substituted withone or more groups including, but not limited to, alkyl, alkynyl,alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy,carboxylic acid, or alkoxy.

I. Crosslinking via Oxidative Coupling

In one aspect described herein is a method for preparing a compound,wherein the method includes reacting a first thiolated compound havingthe formula III

wherein

-   -   Y is a residue of a macromolecule, and    -   L is a polyalkylene group, a polyether group, a polyamide group,        a polyimino group, an aryl group, a polyester, or a        polythioether group, with a second thiolated compound having at        least one SH group in the presence of an oxidant,    -   wherein the first thiolated compound and second thiolated        compound are the same or different compounds.

FIG. 1 depicts one aspect of the method described above for producing afirst thiolated compound having the formula III, where Y is hyaluronan.The first step involves reacting a macromolecule having the formulaY—COOH with the dibydrazide/disulfide compound having the formula A. Thereaction is performed in the presence of a condensing agent. Acondensing agent is any compound that facilitates the reaction betweenthe dihydrazide group of compound A and the COOH group on themacromolecule. In one aspect, the condensing agent is a carbodiimide,including, but not limited to,1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDCI). As depicted inFIG. 1, a mixture of products (B and C) are produced after the firststep. The disulfide bond in compounds B and C is cleaved with a reducingagent. In one aspect, the reducing agent is dithiothreitol. Cleavage ofthe disulfide bonds in compounds B and C produces the first tholatedcompound having the formula III.

The macromolecule is any compound having at least one group that canreact with a hydrazide compound. In one aspect, the macromolecule has atleast one —COOH group or the salt or ester thereof. In another aspect,the macromolecule is an oligonucleotide, a nucleic acid or ametabolically stabilized analogue thereof, a polypeptide, a lipid, aglycoprotein, or a glycolipid. In another aspect, the macromolecule is apolysaccharide, a protein, or a synthetic polymer.

In one aspect, the macromolecule can be a pharmaceutically-acceptablecompound. In one aspect, the pharmaceutically-acceptable compounds caninclude substances capable of preventing an infection systemnically inthe biological system or locally at the defect site, as for example,anti-inflammatory agents such as, but not limited to, pilocarpine,hydrocortisone, prednisolone, cortisone, diclofenac sodium,indomethacin, 6∝-methyl-prednisolone, corticosterone, dexamethasone,prednisone, and the like; antibacterial agents including, but notlimited to, penicillin, cephalosporins, bacitracin, tetracycline,doxycycline, gentamycin, chloroquine, vidarabine, and the like;analgesic agents including, but not limited to, salicylic acid,acetaminophen, ibuprofen, naproxen, piroxicam, flurbiprofen, morphine,and the like; local anesthetics including, but not limited to, cocaine,lidocaine, benzocaine, and the like; immunogens (vaccines) forstimulating antibodies against hepatitis, influenza, measles, rubella,tetanus, polio, rabies, and the like; peptides including, but notlimited to, leuprolide acetate (an LH-RH agonist), nafarelin. and thelike. All compounds are available from Sigma Chemical Co. (Milwaukee,Wis.).

In another aspect, the phannaceutically-acceptable compound can be asubstance or metabolic precursor which is capable of promoting growthand survival of cells and tissues or augmenting the functioning of cellsis useful, as for example, a nerve growth promoting substance such as aganglioside, a nerve growth factor, and the like; a hard or soft tissuegrowth promoting agent such as fibronectin (FN), human growth honnone(HGH), a colony stimulating factor, bone morphogenic protein,platelet-derived growth factor (PDGF), insulin-derived growth factor(IGF-I, IGF-II), transforming growth factor-alpha (TGF-alpha),transforming growth factor-beta (TGF-beta), epiderrnal growth factor(EGF), fibroblast growth factor (FGF), interleukin-1 (IL-1), vascularendothelial growth factor (VEGF) and keratinocyte growth factor (KGF),dried bone material, and the like; and antineoplastic agents such asmethotrexate, 5-fluorouracil, adriamycin, vinblastine, cisplatin,tumor-specific antibodies conjugated to toxins, tumor necrosis factor,and the like.

In another aspect, the pharmaceutically-acceptable compound can includehormones such as progesterone, testosterone, and follicle stimulatinghormone (FSH) (birth control, fertility-enhancement), insulin, and thelike; antihistamines such as diphenhydramine, and the like;cardiovascular agents such as papaverine, streptokinase and the like;anti-ulcer agents such as isopropamide iodide, and the like;bronchodilators such as metaproternal sulfate, aminophylline, and thelike; vasodilators such as theophylline, niacin, minoxidil, and thelike; central nervous system agents such as tranquilizer, B-adrenergicblocking agent, dopamine, and the like; antipsychotic agents such asrisperidone, narcotic antagonists such as naltrexone, naloxone,buprenorphine; and other like substances. All compounds are availablefrom Sigma Chemical Co. (Milwaukee, Wis.).

Polysaccharides useful in the methods described herein have at least onegroup, such as a carboxylic acid group or the salt or ester thereof,that can react with a dihydrazide. In one aspect, the polysaccharide isa glycosaminoglycan (GAG). A GAG is one molecule with many alternatingsubunits. For example, HA is (GlcNAc-GlcUA-)x. Other GAGs are sulfatedat different sugars. Generically, GAGs are represented by the formulaA-B-A-B-A-B, where A is a uronic acid and B is an aminosugar that iseither O— or N-sulfated, where the A and B units can be heterogeneouswith respect to epimeric content or sulfation. Any natural or syntheticpolymer containing uronic acid can be used. In one aspect, Y in formulaIII is a sulfated-GAG.

There are many different types of GAGs, having commonly understoodstructures, which, for example, are within the disclosed compositions,such as chondroitin sulfate, dermatan, heparan, heparin, dermatansulfate, and heparan sulfate. Any GAG known in the art can be used inany of the methods described herein. Glycosaminoglycans can be purchasedfrom Sigma, and many other biochemical suppliers. Alginic acid, pectin,and carboxymethylcellulose are among other carboxylic acid containingpolysaccharides useful in the methods described herein.

In another aspect, the polysaccharide Y in formula III is hyaluronan(HA). HA is a non-sulfated GAG. Hyaluronan is a well known, naturallyoccurring, water soluble polysaccharide composed of two alternativelylinked sugars, D-glucuronic acid and N-acetylglucosamine. The polymer ishydrophilic and highly viscous in aqueous solution at relatively lowsolute concentrations. It often occurs naturally as the sodium salt,sodium hyaluronate. Methods of preparing commercially availablehyaluronan and salts thereof are well known. Hyaluronan can be purchasedfrom Seikagaku Company, Clear Solutions Biotech, Inc., Pharmacia Inc.,Sigma Inc., and many other suppliers. For high molecular weighthyaluronan it is often in the range of 100 to 10,000 disaccharide units.In another aspect, the lower limit of the molecular weight of thehyaluronan is from 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000, or 100,000, and the upper limit is 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or1,000,000, where any of the lower limits can be combined with any of theupper limits. In another aspect, Y in formula III is not hyaluronan.

Y in formula m can also be a synthetic polymer. The synthetic polymerhas at least one carboxylic acid group or the salt or ester thereof,which is capable of reacting with a hydrazide. In one aspect, thesynthetic polymer residue in formula III comprises the synthetic polymercomprises glucuronic acid, polyacrylic acid, polyaspratic acid,polytartaric acid, polyglutamic acid, or polyfumaric acid.

In another aspect, Y in formula III is a protein. Proteins useful in themethods described herein include, but are not limited to, anextracellular matrix protein, a chemically-modified extracellular matrixprotein, or a partially hydrolyzed derivative of an extracellular matrixprotein. The proteins may be naturally occurring or recombinantpolypeptides possessing a cell interactive domain. The protein can alsobe mixtures of proteins, where one or more of the proteins are modified.Specific examples of proteins include, but are not limited to, collagen,elastin, decorin, laminin, or fibronectin.

The identity and length of L in formula III will vary depending upon theend use of the compound. In one aspect, L in formula III is apolyalkylene group. In another aspect, L in formula III is a C₁ to C₂₀polyalkylene group. In another aspect, L in formula I is CH₂CH₂ orCH₂CH₂CH₂. In another aspect, Y is a residue of a polysaccharide orprotein and L is CH₂CH₂ or CH₂CH₂CH₂.

The second thiolated compound is any compound having at least one thiolgroup. The first and second thiolated compounds can be the same ordifferent compounds. In one aspect, the second thiolated compound can beany macromolecule described above. In one aspect, the second thiolatedcompound is a polysaccharide having at least one SH group. Any of thepolysaccharides described above can be used as the second thiolatedcompound. In another aspect, the second thiolated compound comprises asulfated-glycosaminoglycan. In a further aspect, the second thiolatedcompound includes chondroitin sulfate, dermatan, heparan, heparin,dermatan sulfate, heparan sulfate, alginic acid, pectin,carboxymethylcellulose, or hyaluronan having at least one SH gpup.

In another aspect, the second thiolated compound has the formula II

wherein

-   -   Z is a residue of a macromolecule, and    -   L is a polyalkylene group, a polyether group, a polyamide group,        a polyimino group, an aryl group, a polyester, or a        polythioether group.

The macromolecule residue Z can be any of the macromolecules describedabove. In one aspect, the second thiolated compound can be a proteinhaving at least one thiol group. In one aspect, the protein comprises anextracellular matrix protein or a chemically-modified extracellularmatrix protein. In another aspect, the protein comprises collagen,elastin, decorin, laminin, or fibronectin

In another aspect, L in formula II is a polyalkylene group. In anotheraspect, L in formula II is a C₁ to C₂₀ polyalkylene group. In anotheraspect, L in formula II is CH₂CH₂ or CH₂CH₂CH₂. In one aspect, Z is aresidue of hyaluronan and L in formula II is CH₂CH₂ or CH₂CH₂CH₂. In afurther aspect, Z is a residue of gelatin and L in formula II is CH₂CH₂or CH₂CH₂CH₂.

In another aspect, described herein is a method for making a crosslinkedcompound involving reacting

-   -   (a) a first thiolated compound comprising a protein having at        least one SH group; and    -   (b) a second thiolated compound comprising a polysaccharide or        synthetic polymer having at least one SH group, in the presence        of an oxidant.        In this aspect, the first thiolated compound has the formula III        and the second thiolated compound has the formula II        wherein    -   Y is a protein residue;    -   Z is a polysaccharide residue or a residue of a synthetic        polymer; and    -   each L is, independently, a polyalkylene group, a polyether        group, a polyamide group, a polyester group, a polyimino group,        an aryl group, or a polythioether group.        In one aspect, L in formula II and II is, independently, CH₂CH₂        or CH₂CH₂CH₂. In another aspect, Z is a residue of hyaluronan.

The reaction between the first and second thiolated compounds isperformed in the presence of an oxidant. In one aspect, the reactionbetween the first and second thiolated compounds can be conducted in thepresence of any gas that contains oxygen. In one aspect, the oxidant isair. This aspect also contemplates the addition of a second oxidant toexpedite the reaction. In another aspect, the reaction can be performedunder an inert atmosphere (i.e., oxygen free), and an oxidant is addedto the reaction. Examples of oxidants useful in this method include, butare not limited to, molecular iodine, hydrogen peroxide, alkylhydroperoxides, peroxy acids, dialkyl sulfoxides, high valent metalssuch as Co⁺³ and Ce⁺⁴, metal oxides of manganese, lead, and chromium,and halogen transfer agents. The oxidants disclosed in Capozzi, G.;Modena, G. In The Chemistry of the Thiol Group Part II; Patai, S., Ed.;Wiley: New York, 1974; pp 785-839, which is incorporated by reference inits entirety, are useful in the methods described herein.

The reaction between the first and second thiolated compounds can beconducted in a buffer solution that is slightly basic. The amount of thefirst thiolated compound relative the amount of the second thiolatedcompound can vary. In one aspect, the volume ratio of the firstthiolated compound to the second thiolated compound is from 99:1, 90:10,80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, or 1:99. In oneaspect, the first and second thiolated compound react in air and areallowed to dry at room temperature. In this aspect, the dried materialcan be exposed to a second oxidant, such as hydrogen peroxide. Theresultant compound can then be rinsed with water to remove any unreactedfirst and/or second thiolated compound and any unused oxidant. Oneadvantage of preparing coupled compound via the oxidative couplingmethodology described herein is that crosslinking can occur in anaqueous media under physiologically benign conditions without thenecessity of additional crosslink-ing reagents.

The compounds produced using the methods described above have at leastone fragment comprising the formula VI

wherein

-   -   Y is a residue of a macromolecule, wherein Y is not a residue of        hyaluronan; and    -   G is a residue of a thiolated compound.

The term “fragment” as used herein refers to the entire molecule itselfor a portion or segment of a larger molecule. For example, Y in formulaVI may be high molecular weight polysaccharide that is crosslinked bydisulfide linkage with another polysaccharide, synthetic polymer, orthiolated polymer to produce the coupled compound. Alternatively, thecoupled compound may have multiple disulfide linkages. The compound hasat a minimum one unit depicted in formula VI, which represents at leastone disulfide linkage as the result of at least one first thiolatedcompound that reacted with at least one second thiolated compound viaoxidation.

The macromolecule (Y) and thiolated compound (G) can be any of themacromolecules described above. In one aspect, Y in formula VI is apolysaccharide, a protein, or a synthetic polymer. In another aspect,the fragment comprises the formula VIII

wherein

-   -   Y is a residue of a macromolecule, wherein Y is not a residue of        hyaluronan;    -   L is a polyalkylene group, a polyether group, a polyamide group,        a polyimino group, an aryl group, a polyester, or a        polythioether group; and    -   G is a residue of a thiolated compound.

In one aspect, Y in formula VIII is a residue of any of theglycosaminoglycans described above including, but not limited to,chondroitin sulfate, dermatan, heparan, heparin, dermatan sulfate,heparan sulfate, alginic acid, pectin, or carboxymethylcellulose. In afurther aspect, L in formula VIII is CH₂CH₂ or CH₂CH₂CH₂. In anotheraspect, G is a residue of any of the polysaccharides described above,including a glycosaminoglycan such as chondroitin sulfate, dermatan,heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid,pectin, carboxymethylcellulose, or hyaluronan.

II. Coupling Compounds via the Reaction Between a Thiol Compound and aThiol-Reactive Compound

In another aspect, described herein is a method for coupling two or morecompounds by reacting a first thiolated macromolecule having at leastone SH group with at least one compound having at one thiol-reactiveelectrophilic functional group. In one aspect, the compound has at leasttwo-thiol reactive functional groups.

Any of the macromolecules described above can be used in this aspect.Two or more different macromolecules can be used in this method. Forexample, a second thiolated macromolecule can be used in combinationwith the first thiolated macromolecule. In this aspect, the first andsecond thiolated macromolecule can be the same or different compounds.

In one aspect, the macromolecule is a polysaccharide. In this aspect,the polysaccharide is a sulfated-glycosaminoglycan including, but notlimited to, chondroitin sulfate, dermatan, heparan, heparin, dermatansulfate, heparan sulfate, alginic acid, pectin, orcarboxymethylcellulose. In another aspect, the polysaccharide ishyaluronan. In another aspect, the polysaccharide has the formula IIIdescribed above. In this aspect, Y is a residue of hyaluronan and L isCH₂CH₂ or CH₂CH₂CH₂.

In another aspect, the macromolecule is a compound having the formulaIII, wherein Y is a protein. Any of the proteins described above can beused in this aspect. In one aspect, the protein is collagen, elastin,decorin, laminin, or fibronectin.

A compound having at least one thiol-reactive electrophilic group isalso used in this aspect of the method. The term “thiol-reactiveelectrophilic group” as used herein is any group that is susceptible tonucleophilic attack by the lone-pair electrons on the sulfur atom of thethiol group or by the thiolate anion. Examples of thiol-reactiveelectrophilic groups include groups that have good leaving groups. Forexample, an alkyl group having a halide or alkoxy group attached to itor an α-halocarbonyl group are examples of thiol-reactive electrophilicgroups. In another aspect, the thiol-reactive electrophilic group is anelectron-deficient vinyl group. The term “an electron-deficient vinylgroup” as used herein is a group having a carbon-carbon double bond andan electron-withdrawing group attached to one of the carbon atoms. Anelectron-deficient vinyl group is depicted in the formula C_(β)═C_(α)X,where X is the electron-withdrawing group. When the electron-withdrawinggroup is attached to Cα, the other carbon atom of the vinyl group (Cβ)is more susceptible to nucleophilic attack by the thiol group. This typeof addition to an activated carbon-carbon double bond is referred to asa Michael addition. Examples of electron-withdrawing groups include, butare not limited to, a nitro group, a cyano group, an ester group, analdehyde group, a keto group, a sulfone group, or an amide group.Examples of compounds possessing thiol-reactive electrphilic groupsinclude, but are not limited to, maleimides, vinyl sulfones.acrylonitriles, α-methylene esters, quinone methides, acryloyl esters oramides, or α-halo esters or amides.

In one aspect, the thiol-reactive compound has two electron-deficientvinyl groups, wherein the two electron-deficient vinyl groups are thesame. In another aspect, the thiol-reactive compound is a diacrylate, adimethacrylate, a diacrylamide, a dimethacrylamide, or a combinationthereof.

In another aspect, the thiol-reactive compound has the formula V

wherein

-   -   R³ and R⁴ are, independently, hydrogen or lower alkyl;    -   U and V are, independently, O or NR⁵, wherein R⁵ is,        independently, hydrogen or lower alkyl; and    -   M is a polyalkylene group, a polyether group, a polyamide group,        a polyimino group, a polyester, an aryl group, or a        polythioether group.

In one aspect, R³ and R⁴ are hydrogen, U and V are oxygen, and M is apolyether group. In another aspect, R³ and R⁴ are hydrogen, U and V areNH, and M is a polyether group. In a further aspect, R³ and R⁴ aremethyl, U and V are oxygen, and M is a polyether group. In anotheraspect, R³ and R⁴ are methyl, U and V are NH, and M is a polyethergroup.

In another aspect, the thiol-reactive compound is any ofpharmaceutically-acceptable compounds described above containing atleast one thiol-reactive electrophilic group. FIG. 22 depicts oneembodiment of this aspect. Mitomycin C (MMC) is converted to thecorresponding acrylate (MMC-acrylate). MMC-acrylate is then coupled withthe hydrazide-modified hyaluronan thiol compound HA-DTPH to produceHA-DT H-MMC. HA-DT H-MMC contains one or more free thiols groups, whichthen can couple with PEGDA to produce HA-DTPH-PEGDA-MMC, which isdepicted in FIG. 23.

In another aspect, the first thiolated macromolecule has the formula IIIdescribed above, wherein Y is a residue of polysaccharide, and L isCH₂CH₂ or CH₂CH₂CH₂, and the thiol-reactive compound has the formula Vdescribed above, wherein R³ and R⁴ are, independently, hydrogen or loweralkyl; U and V are, independently, O or NR⁵, wherein R⁵ is,independently, hydrogen or lower alkyl; and M is a polyether group. Inthis aspect, (1) Y is a residue of hyaluronan, and the reaction furthercomprises reacting gelatin having at least one thiol group with thecompound having the formula V; (2) the polysaccharide includes a firstpolysaccharide and second polysaccharide having the formula I, whereinin the first polysaccharide, Y is a residue of a firstsulfated-glycosaminoglycan, and in the second polysaccharide, Y is aresidue of a second sulfated-glycosaminoglycan, wherein the first andsecond sulfated-glycosaminoglycans are the same or different; (3) thepolysaccharide includes a first polysaccharide and second polysaccharidehaving the formula I, wherein in the first polysaccharide, Y is aresidue of hyaluronan, and in the second polysaccharide, Y is a residueof a sulfated-glycosaminoglycan; or (4) further reacting a protein, anextracellular matrix protein, or growth factor having at least one thiolgroup with the compound having the formula V.

In another aspect, described herein is a method for coupling a compoundby reacting a first thiolated macromolecule having at least onethiol-reactive electrophilic functional group with at least one compoundhaving at least two thiol groups.

Any of the macromolecules and thiol-reactive electrophilic functionalgroups described above can be used in this aspect. In this aspect, thefirst thiolated macromolecule having at least one thiol-reactiveelectrophilic functional group and the thiolated compound have theformula I

wherein

-   -   Y is a residue of the macromolecule;    -   Q is the thiol-reactive electrophilic functional group or an SH        group; and    -   L is a polyalkylene group, a polyether group, a polyamide group,        a polyimino group, a polyester, an aryl group, or a        polythioether group.        In one aspect, when Q is thiol-reactive electrophilic functional        group, Y is hyaluronan and L is CH₂CH₂ or CH₂CH₂CH₂. In another        aspect, Q is an acrylate, a methacrylate, an acrylamide, or a        methacrylamide.

In one aspect, examples of compounds having at least two thiol groupsinclude, but are not limited to, propane-1,3-dithiol, polyethyleneglycol-α,Ω-dithiol, para, ortho, or meta-bisbenzyl thiol,dithiothreitol, a peptide containing two or more cysteine residues, ordendrimeric thiols.

The compounds produced by coupling a thiolated compound with a compoundhaving at least one thiol-reactive electrophilic functional grouppossess at least one fragment of the formula VII

wherein

-   -   R⁷ and R⁸ are, independently, hydrogen or lower alkyl;    -   X is an electron-withdrawing group; and    -   Y is a residue of a macromolecule.        In this aspect, X and Y in formula VII can be any of the        electron-withdrawing groups and macromolecules, respectively,        described above. In one aspect, Y is a residue of a        polysaccharide such as hyaluronan or a        sulfated-glycosaminoglycan. In another aspect, R⁷ is hydrogen        and R⁸ is hydrogen or methyl. In another aspect, Y is a residue        of hyaluronan or a sulfated-glycosaminoglycan; R⁷ is hydrogen;        R⁸ is hydrogen or methyl; and X is an ester group or an amide        group.

In one aspect, the fragment has the formula IV

wherein

-   -   R³ and R⁴ are, independently, hydrogen or lower alkyl;    -   U and V are, independently, O or NR⁵, wherein R⁵ is,        independently, hydrogen or lower alkyl;    -   Y is a residue of a protein;    -   Z is a polysaccharide residue or a residue of synthetic polymer,        and    -   M is a polyalkylene group, a polyether group, a polyamide group,        a polyester group, a polyimino group, an aryl group, or a        polythioether group.

In one aspect, Y in formula IV has the formula IX

wherein

-   -   Y′ is a residue of a protein, where the protein is any of the        proteins described above;    -   L is a polyalkylene group, a polyether group, a polyamide group,        a polyester group, a polyimino group, an aryl group, or a        polythioether group, wherein the L group is covalently bonded to        the sulfur atom.

In one aspect, Z in formula IV has the formula X

wherein

-   -   Z′ is a polysaccharide residue or a residue of a synthetic        polymer; and    -   L is a polyalkylene group, a polyether group, a polyamide group,        a polyester group, a polyimino group, an aryl group, or a        polythioether group, wherein the L group is covalently bonded to        the sulfur atom.

In one aspect, the reaction between the thiol reactive compound andthiol compound is generally conducted at a pH of from 7 to 12, 7.5 to11, 7.5 to 10, or 7.5 to 9.5, or a pH of 8. In one aspect, the solventused can be water (alone) or an aqueous containing organic solvent. Inone aspect, when the mixed solvent system is used, a base such as aprimary, secondary, or tertiary amine can used. In one aspect, an excessof thiol compound is used relative to the thiol-reactive compound inorder to ensure that all of the thiol-reactive compound is consumedduring the reaction. Depending upon the selection of the thiol reactivecompound, the thiol compound, the pH of the reaction, and the solventselected, coupling can occur from within minutes to several days. If thereaction is performed in the presence of an oxidant, such as air, thethiol compound can react with itself or another thiol compound viaoxidative addition to form a disulfide linkage in addition to reactingwith the thiol-reactive compound.

III. Crosslinked Proteins

Described herein are methods for coupling a protein with anothermolecule using hydrazide compounds. In one aspect, a protein having atleast one hydrazide-reactive group is reacted with a compound having atleast one hydrazide group. In another aspect, a protein having at leastone hydrazide group is reacted with a compound having at least onehydrazide-reactive group. In one aspect, the hydrazide-reactive groupcan be a —COOH group (or the salt or ester thereof), an aldehyde group,or a ketone group. The techniques disclosed in international publicationnos. WO 02106373 A1 and WO 02/090390 A1, which are incorporated byreference in their entireties, can be used in this aspect

In one aspect, the coupled protein has the formula XI

wherein

-   -   J comprises a protein residue; and    -   E comprises a fluorescent tag, a radiolabel, a targeting moiety,        a lipid, a peptide, a radionuclide chelator with a radionuclide,        a spin-label, a PEG camouflage, a metal surface, a glass        surface, a plastic surface, or a combination thereof.

The protein residue can be any protein that has at least onehydrazide-reactive group or at least one hydrazide group. In one aspect,the protein can be an extracellular matrix protein, a partiallyhydrolyzed extracellular matrix protein, or a chernically-modifledextracellular matrix protein. In another aspect, the protein iscollagen, elastin, decorin, laminin, or fibronectin.

In one aspect, E in formula XI is a reporter group. Examples of reportergroups include, but are not limited to, a fluorescent tag, a radiolabel,a targeting moiety, a lipid, a peptide, a radionuclide chelator with aradionuclide, a spin-label, a PEG camouflage, a glass surface, a plasticsurface, or a combination thereof. Examples of hydrazide-modifiedfluorescent groups include, but are not limited to, BODIPY-hydrazide,fluorescein hydrazide, or NBD-hexanoyl -hydrazide. Examples ofhydrazide-modified radiolabels include, but are not limited to,125I-tyrosine-hydrazide, 3H-acetyl-hydrazide, or ¹⁴C-acetyl-hydrazide.Examples of hydrazide-modified targeting moieties include, but are notlimited to, 6-aminohexanoylhydrazide (Z) of integrin targeting peptide,such as ZYRGDS, Z-tat decapeptide for cell penetration, Z-GFLG forlysosome targeting, HA oligosaccharide hydrazide for CD-44 cancertargeting, or N-Ac glucosamine derivative for liver targeting. Examplesof hydrazide-modified lipids include, but are not limited to, hydrazideof 2′-succinate of Taxol or 2′succinate of a glucocorticosteroids,alkanoic or perfluoroalkanoate hydrazides, phosphatidylserine hydrazide,or cholic acid hydrazide. Examples of hydrazide-modified radionuclidesinclude, but are not limited to, the reaction product between DTPAanhydride and hydrazine to produce the corresponding hydrazide, couplingthe hydrazide to a protein, then adding a nuclide such as In-111,Tc-99m, or Y-90. Examples of spin labels include, but are not limitedto, proxyl or doxyl groups. Examples of glass surfaces include, but arenot limited to, glass silanized with an epoxy or activated ester or athiol-reactive electrophilic functional group, beads, or coverslips.Examples of plastics include, but are not limited to, plasma-etchedpolypropylene, chemically-modified polystyrene with hydrazide, or anyother plastic material. In another aspect, E is a crosslinkable thiolreactive-electrophilic groups such, but not limited to, acrylichydrazide or methacrylic hydrazide.

In another aspect, described herein is a kit including (1) a compoundhaving at least one hydrazide group; (2) a condensing agent; (3) abuffer reagent; and (4) a purification column. In one aspect, thecompound can be any compound having at least one hydrazide group and atleast one of the reporter groups described above. Use of the kitgenerally involves admixing components (1)-(3) together with a proteinhaving at least one hydrazide-reactive group. Components (1)-(3) and theprotein can be added in any order. After the protein and the compoundhaving at least one hyrazide group have reacted with one another toproduce the coupled protein, the coupled protein is then purified bypassing the admixture containing the coupled protein through apurification column. Purification columns and techniques for using thesame are known in the art.

IV. Pharmaceutical Compositions

In one aspect, any of the compounds produced by the methods describedabove can include at least one pharmaceutically-acceptable compound. Theresulting pharmaceutical composition can provide a system for sustained,continuous delivery of drugs and other biologically-active agents totissues adjacent to or distant from the application site. Thebiologically-active agent is capable of providing a local or systemicbiological, physiological or therapeutic effect in the biological systemto which it is applied. For example, the agent can act to controlinfection or inflammation, enhance cell growth and tissue regeneration,control tumor growth, act as an analgesic, promote anti-cell attachment,and enhance bone growth, among other functions. Additionally, any of thecompounds described herein can contain combinations of two or morepharmaceutically-acceptable compounds.

In one aspect, the pharmaceutically-acceptable compounds can includesubstances capable of preventing an infection systemically in thebiological system or locally at the defect site, as for example,anti-inflammatory agents such as, but not limited to, pilocarpine,hydrocortisone, prednisolone, cortisone, diclofenac sodium,indomethacin, 6∝-methyl-prednisolone, corticosterone, dexamethasone,prednisone, and the like; antibacterial agents including, but notlimited to, penicillin, cephalosporins, bacitracin, tetracycline,doxycycline, gentamycin, chloroquine, vidarabine, and the like;analgesic agents including, but not limited to, salicylic acid,acetaminophen, ibuprofen, naproxen, piroxicam, flurbiprofen, morphine,and the like; local anesthetics including, but not limited to, cocaine,lidocaine, benzocaine, and the like; immunogens (vaccines) forstimulating antibodies against hepatitis, influenza, measles, rubella,tetanus, polio, rabies, and the like; peptides including, but notlimited to, leuprolide acetate (an LH-RH agonist), nafarelin, and thelike. All compounds are available from Sigma Chemical Co. (Milwaukee,Wis.).

Additionally, a substance or metabolic precursor which is capable ofpromoting growth and survival of cells and tissues or augmenting thefunctioning of cells is useful, as for example, a nerve growth promotingsubstance such as a ganglioside, a nerve growth factor, and the like; ahard or soft tissue growth promoting agent such as fibronectin (FN),human growth hormone (HGH), a colony stimulating factor, bonemorphogenic protein, platelet-derived growth factor (PDGF),insulin-derived growth factor (IGF-I, IGF-II), transforming growthfactor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta),epidermal growth factor (EGF), fibroblast growth factor (FGF),interleukin-1 (IL-1), vascular endothelial growth factor (VEGF) andkeratinocyte growth factor (KGF), dried bone material, and the like; andantineoplastic agents such as methotrexate, 5-fluorouracil, adriamycin,vinblastine, cisplatin, tumor-specific antibodies conjugated to toxins,tumor necrosis factor, and the like.

Other useful substances include hormones such as progesterone,testosterone, and follicle stimulating hormone (FSH) (birth control,fertility-enhancement), insulin, and the like; antihistamines such asdiphenhydramine, and the like; cardiovascular agents such as papaverine,streptokinase and the like; anti-ulcer agents such as isopropamideiodide, and the like; bronchodilators such as metaproternal sulfate;aminophylline, and the like; vasodilators such as theophylline, niacin,minoxidil, and the like; central nervous system agents such astranquilizer, B-adrenergic blocking agent, dopamine, and the like;antipsychotic agents such as risperidone, narcotic antagonists such asnaltrexone, naloxone, buprenorphine; and other like substances. Allcompounds are available from Sigrna Chemical Co. (Milwaukee, Wis.).

The pharmaceutical compositions can be prepared using techniques knownin the art. In one aspect, the composition is prepared by admixing acompound described herein with a pharmaceutically-acceptable compound.The term “admixing” is defined as mixing the two components together sothat there is no chemical reaction or physical interaction. The term“admixing” also includes the chemical reaction or physical interactionbetween the compound and the pharmaceutically-acceptable compound.Covalent bonding to reactive therapeutic drugs, e.g., those havingreactive carboxyl groups, can be undertaken on the compound. Forexample, first, carboxylate-containing chemicals such asanti-inflammatory drugs ibuprofen or hydrocortisone-hemisuccinate can beconverted to the corresponding N-hydroxysuccinimide (NHS) active estersand can further react with the NH₂ group of the dihydrazide-modifiedpolysaccharide. Second, non-covalent entrapment of a pharmacologicallyactive agent in a cross-linked polysaccharide is also possible. Third,electrostatic or hydrophobic interactions can facilitate retention of apharmaceutically-acceptable compound in a modified: polysaccharide. Forexample, the hydrazido group can non-covalently interact, e.g., withcarboxylic acid-containing steroids and their analogs, andanti-inflammatory drugs such as Ibuprofen (2-(4-iso-butylphenyl)propionic acid). The protonated hydrazido group can form salts with awide variety of anionic materials such as proteins, heparin or dermatansulfates, oligonucleotides, phosphate esters, and the like.

It will be appreciated that the actual preferred amounts of activecompound in a specified case will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, and the particular situs and subject being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g. by customary comparison of the differentialactivities of the subject compounds and of a known agent, e.g., by meansof an appropriate conventional pharmacological protocol. Physicians andformulators, skilled in the art of determining doses of pharmaceuticalcompounds, will have no problems determining dose according to standardrecommendations (Physicians Desk Reference, Barnhart Publishing (1999).

Pharmaceutical compositions described herein can be formulated in anyexcipient the biological system or entity can tolerate. Examples of suchexcipients include, but are not limited to, water, saline, Ringer'ssolution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, vegetable oils such as olive oil and sesame oil,triglycerides, propylene glycol, polyethylene glycol, and injectableorganic esters such as ethyl oleate can also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosol, cresols, formalin and benzyl alcohol.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration to humans,including solutions such as sterile water, saline, and bufferedsolutions at physiological pH.

Molecules intended for pharmaceutical delivery can be formulated in apharmaceutical composition. Pharmaceutical compositions can includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more activeingredients such as antimicrobial agents, antiinflammatory agents,anesthetics, and the like.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration can be topically (includingophthalmically, vaginally, rectally, intranasally).

Preparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles, if needed forcollateral use of the disclosed compositions and methods, include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles, if needed forcollateral use of the disclosed compositions and methods, include fluidand nutrient replenishers, electrolyte replenishers (such as those basedon Ringer's dextrose), and the like. Preservatives and other additivescan also be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable.

Dosing is dependent on severity and responsiveness of the condition to,be treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until one ofordinary skill in the art determines the delivery should cease. Personsof ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates.

In one aspect, any of the compounds and pharmaceutical compositions caninclude living cells. Examples of living cells include, but are notlimited to, fibroblasts, hepatocytes, chondrocytes, stem cells, bonemarrow, muscle cells, cardiac myocytes, neuronal cells, or pancreaticislet cells.

V. Methods of Use

The compounds and pharmaceutical compositions described herein can beused for a variety of uses related to drug delivery, small moleculedelivery, wound healing, burn injury healing, and tissue regeneration.The disclosed compositions are useful for situations which benefit froma hydrated, pericellular environment in which assembly of other matrixcomponents, presentation of growth and differentiation factors, cellmigration, or tissue regeneration are desirable.

The compounds and pharmaceutical compositions described herein can beplaced directly in or on any biological system without purification asit is composed of biocompatible materials. Examples of sites thecompounds can be placed include, but not limited to, soft tissue such asmuscle or fat; hard tissue such as bone or cartilage: areas of tissueregeneration; a void space such as periodontal pocket; surgical incisionor other formed pocket or cavity; a natural cavity such as the oral,vaginal, rectal or nasal cavities, the cul-de-sac of the eye, and thelike; the peritoneal cavity and organs contained within, and other sitesinto or onto which the compounds can be placed including a skin surfacedefect such as a cut, scrape or burn area. The present compounds can bebiodegradeable and naturally occurring enzymes will act to degrade themover time. Components of the compound can be “bioabsorbable” in that thecomponents of the compound will be broken down and absorbed within thebiological system, for example, by a cell, tissue and the like.Additionally, the compounds, especially compounds that have not beenrehydrated, can be applied to a biological system to absorb fluid froman area of interest.

The compounds described herein can be used as a carrier for a widevariety of releasable biologically active substances having curative ortherapeutic value for human or non-human animals. Many of thesesubstances which can be carried by the compound are discussed above.Included among biologically active materials which are suitable forincorporation into the gels of the invention are therapeutic drugs,e.g., anti-inflammatory agents, anti-pyretic agents, steroidal andnon-steroidal drugs for anti-inflammatory use, hormones, growth factors,contraceptive agents, antivirals, antibacterials, antifungals,analgesics, hypnotics, sedatives, tranquilizers, anti-convulsants,muscle relaxants, local anesthetics, antispasmodics, antiulcer drugs,peptidic agonists, sympathiomimetic agents, cardiovascular agents,antitumor agents, oligonucleotides and their analogues and so forth. Abiologically active substance is added in pharmaceutically activeamounts.

In one aspect, the compounds and compositions described herein can beused for the delivery of living cells to a subject. Any of the livingcells described above can be used in the aspect.

In one aspect, the compounds and compositions can be used for thedelivery of growth factors and molecules related to growth factors. Forexample the growth factors can be a nerve growth promoting substancesuch as a ganglioside, a nerve growth factor, and the like; a hard orsoft tissue growth promoting agent such as fibronectin (FN), humrangrowth hormone (HGH), a colony stimulating factor, bone morphogenicprotein, platelet-derived growth factor (PDGF), insulin derived growthfactor (IGF-I, IGF-II), transforming growth factor-alpha (TGF-alpha),transforming growth factor-beta (TGF-beta), epidermal growth factor(EGF), fibroblast growth factor (FGF), interleukin-1 (IL-1). Preferredgrowth factors are bFGF and TGF-β. Also preferred are vascularendothelial growth factor (VEGF) and keratinocyte growth factor (KGF).

In another aspect, anti-inflammatories bearing carboxyl groups such asibuprofen, naproxen, ketoprofen and indomethacin can be used. Otherbiologically active substances are peptides, which are naturallyoccurring, non-naturally occurring or synthetic polypeptides or theirisosteres, such as small peptide hormones or hormone analogues andprotease inhibitors. Spermicides, antibacterials, antivirals,antifungals and antiproliferatives such as fluorodeoxyuracil andadriamycin can also be used. These substances are all known in the art.Compounds are available from Sigma Chemical Company (St. Louis, Mo.).

The term “therapeutic drugs” as used herein is intended to include thosedefined in the Federal Food, Drug and Cosmetic Act. The United StatesPharmacopeia (USP) and the National Formulary (NF) are the recognizedstandards for potency and purity for most common drug products.

In one aspect, the pharmaceutically acceptable compound is pilocarpine,hydrocortisone, prednisolone, cortisone, diclofenac sodium,indomethacin, 6∝-methyl-prednisolone, corticosterone, dexamethasone andprednisone. However, methods are also provided wherein delivery of apharmaceutically-acceptable compound is for a medical purpose selectedfrom the group of delivery of contraceptive agents, treatingpostsurgical adhesions, promoting skin growth, preventing scarring,dressing wounds, conducting viscosurgery, conductingviscosupplementation, engineering tissue.

The rate of drug delivery depends on the hydrophobicity of the moleculebeing released. Hydrophobic molecules, such as dexamethazone andprednisone are released slowly from the compound as it swells in anaqueous environment, while hydrophilic molecules, such as pilocarpine,hydrocortisone, prednisolone, cortisone, diclofenac sodium,indomethacin, 6∝-methyl-prednisolone and corticosterone, are releasedquickly. The ability of the compound to maintain a slow, sustainedrelease of steroidal anti-inflammatories makes the compounds describedherein extremely useful for wound healing after trauma or surgicalintervention. Additionally, the compound can be used as a barrier systemfor enhancing cell growth and tissue regeneration.

In certain methods the delivery of molecules or reagents related toangiogenesis and vascularization are achieved. Disclosed are methods fordelivering agents, such as VEGF, that stimulate microvascularization.Also disclosed are methods for the delivery of agents that can inhibitangiogenesis and vascularization, such as those compounds and reagentsuseful for this purpose disclosed in but not limited to U.S. Pat. No.6,174,861 for “Methods of inhibiting angiogenesis via increasing in vivoconcentrations of endostatin protein;” U.S. Pat. No. 6,086,865 for“Methods of treating angiogenesis-induced diseases and pharmaceuticalcompositions thereof;” U.S. Pat. No. 6,024,688 for “Angiostatinfragments and method of use;” U.S. Pat. No. 6,017,954 for “Method oftreating tumors using O-substituted fumagillol derivatives;” U.S. Pat.No. 5,945,403 for “Angiostatin fragments and method of use;” U.S. Pat.No. 5,892,069 “Estrogenic compounds as anti-mitotic agents;” for U.S.Pat. No. 5,885,795 for “Methods of expressing angiostatic protein;” U.S.Pat. No. 5,861,372 for “Aggregate angiostatin and method of use;” U.S.Pat. No. 5,854,221 for “Endothelial cell proliferation inhibitor andmethod of use;” U.S. Pat. No. 5,854,205 for “Therapeutic antiangiogeniccompositions and methods;” U.S. Pat. No. 5,837,682 for “Angiostatinfragments and method of use;” U.S. Pat. No. 5,792,845 for “Nucleotidesencoding angiostatin protein and method of use;” U.S. Pat. No. 5,733,876for “Method of inhibiting angiogenesis;” U.S. Pat. No. 5,698,586 for“Angiogenesis inhibitory agent;” U.S. Pat. No. 5,661,143 for “Estrogeniccompounds as anti-mitotic agents;” U.S. Pat. No. 5,639,725 for“Angiostatin protein;” U.S. Pat. No. 5,504,074 for “Estrogenic compoundsas anti-angiogenic agents;” U.S. Pat. No. 5,290,807 for “Method forregressing angiogenesis using o-substituted fumagillol derivatives;” andU.S. Pat. No. 5,135,919 for “Method and a pharmaceutical composition forthe inhibition of angiogenesis” which are herein incorporated byreference for the material related to molecules for angiogenesisinhibition.

Described herein are methods for improving wound healing in a subject inneed of such improvement by contacting any of the compounds orpharmaceutical compositions described herein with a wound of a subjectin need of wound healing improvement. Also provided are methods todeliver at least one pharmaceutically-acceptable compound to a patientin need of such delivery by contacting any of the compounds orpharmaceutical compositions described herein with at least one tissuecapable of receiving said pharmaceutically-acceptable compound.

The disclosed compositions can be used for treating a wide variety oftissue defects in an animal, for example, a tissue with a void such as aperiodontal pocket, a shallow or deep cutaneous wound, a surgicalincision, a bone or cartilage defect, and the like. For example, thecompounds described herein can be in the form of a hydrogel film. Thehydrogel film can be applied to a defect in bone tissue such as afracture in an arm or leg bone, a defect in a tooth, a cartilage defectin the joint, ear, nose, or throat, and the like. The hydrogel filmcomposed of the compound described herein can also function as a barriersystem for guided tissue regeneration by providing a surface on orthrough which the cells can grow. To enhance regeneration of a hardtissue such as bone tissue, it is preferred that the hydrogel filmprovides support for new cell growth that will replace the matrix as itbecomes gradually absorbed or eroded by body fluids.

The hydrogel film composed of a compound described herein can bedelivered onto cells, tissues, and/or organs, for example, by injection,spraying, squirting, brushing, painting, coating, and the like. Deliverycan also be via a cannula, catheter, syringe with or without a needle,pressure applicator, pump, and the like. The compound can be appliedonto a tissue in the form of a film, for example, to provide a filmdressing on the surface of the tissue, and/or to adhere to a tissue toanother tissue or hydrogel film, among other applications.

In one aspect, the compounds described herein are administered viainjection. For many clinical uses, when the compound is in the form of ahydrogel film, injectable hydrogels are preferred for three mainreasons. First, an injectable hydrogel could be formed into any desiredshape at the site of injury. Because the initial hydrogels can be solsor moldable putties, the systems can be positioned in complex shapes andthen subsequently crosslinked to conform to the required dimensions.Second, the hydrogel would adhere to the tissue during gel formation,and the resulting mechanical interlocking arising from surfacemicroroughness would strengthen the tissue-hydrogel interface. Third,introduction of an in situ-crosslinkable hydrogel could be accomplishedusing needle or by laparoscopic methods, thereby minimizing theinvasiveness of the surgical technique.

The compounds described herein can be used to treat periodontal disease,gingival tissue overlying the root of the tooth can be excised to forman envelope or pocket, and the composition delivered into the pocket andagainst the exposed root. The compounds can also be delivered to a toothdefect by making an incision through the gingival tissue to expose theroot, and then applying the material through the incision onto the rootsurface by placing, brushing, squirting, or other means.

When used to treat a defect on skin or other tissue, the compoundsdescribed herein can be in the form of a hydrogel film that can beplaced on top of the desired area. In this aspect, the hydrogel film ismalleable and can be manipulated to conform to the contours of thetissue defect.

The compounds described herein can be applied to an implantable devicesuch as a suture, claps, prosthesis, catheter, metal screw, bone plate,pin, a bandage such as gauze, and the like, to enhance the compatibilityand/or performance or function of an implantable device with a bodytissue in an implant site. The compounds can be used to coat theimplantable device. For example, the compounds could be used to coat therough surface of an implantable device to enhance the compatibility ofthe device by providing a biocompatable smooth surface which reduces theoccurrence of abrasions from the contact of rough edges with theadjacent tissue. The compounds can also be used to enhance theperformance or function of an implantable device. For example, when thecompound is a hydrogel film, the hydrogel film can be applied to a gauzebandage to enhance its compatibility or adhesion with the tissue towhich it is applied. The hydrogel film can also be applied around adevice such as a catheter or colostomy that is inserted through anincision into the body to help secure the catheter/colosotomy in placeand/or to fill the void between the device and tissue and form a tightseal to reduce bacterial infection and loss of body fluid.

It is understood that the disclosed compositions and methods can beapplied to a subject in need of tissue regeneration. For example, cellscan be incorporated into the compounds described herein forimplantation. In one aspect the subject is a mammal. Preferred mammalsto which the compositions and methods apply are mice, rats, cows orcattle, horses, sheep, goats, cats, dogs, and primates, including apes,chimpanzees, orangatangs, and humans. In another aspect, the compoundsand compositions described herein can be applied to birds.

When being used in areas related to tissue regeneration such as wound orburn healing, it is not necessary that the disclosed methods andcompositions eliminate the need for one or more related acceptedtherapies. It is understood that any decrease in the length of time forrecovery or increase in the quality of the recovery obtained by therecipient of the disclosed compositions or methods has obtained somebenefit. It is also understood that some of the disclosed compositionsand methods can be used to prevent or reduce fibrotic adhesionsoccurring as a result of wound closure as a result of trauma, suchsurgery. It is also understood that collateral affects provided by thedisclosed compositions and compounds are desirable but not required,such as improved bacterial resistance or reduced pain etc.

It is understood that any given particular aspect of the disclosedcompositions and methods can be easily compared to the specific examplesand embodiments disclosed herein, including the non-polysaccharide basedreagents discussed in the Examples. By performing such a comparison, therelative efficacy of each particular embodiment can be easilydetermined. Particularly preferred assays for the various uses are thoseassays which are disclosed in the Examples herein, and it is understoodthat these assays, while not necessarily limiting, can be performed withany of the compositions and methods disclosed herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated,: and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

I. Materials

Fermentation-derived hyaluronan (HA, sodium salt, M_(w) 1.5 MDa) wasobtained from Clear Solutions Biotech, Inc. (Stony Brook, N.Y.).1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI),3,3′-dithiobis(propanoic acid), 4,4-dithiobis(butanoic acid), andpoly(ethylene glycol) acrylate (M_(w) 375), and hydrazine hydrate werefrom Aldrich Chemical Co. (Milwaukee, Wis.). Dulbecco's phosphatebuffered saline (PBS), bovine testicular hyaluronidase (HAse, 330 U/mg)and blue dextran (M_(w) 200,000) was from Sigma Chemical Co. (St. Louis,Mo.). Dithiothreitol (DTT) was from Diagnostic Chemicals Limited(Oxford, Conn.). 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB) was fromAcros (Houston, Tex.). 3,3′-dithiobis(propanoic dihydrazide) (DTP) and4,4′-dithiobis(butyric dihydrazide) (DTB) was synthesized as previouslydescribed in Vercruysse K P, Marecak D M, Marecek J F, and Prestwich GD. “Synthesis and in vitro degradation of new polyvalent hydrazidecross-linked hydrogels of hyaluronic acid,” Bioconjugate Chem1997;8:686-694, which is incorporated by reference in its entirety.Poly(ethylene glycol)-diacrylate (PEGDA), poly(ethyleneglycol)-dimethacrylate (PEGDM), poly(ethylene glycol)-diacrylamide(PEGDAA) and poly(ethylene glycol)-dimethacrylamide (PEGDMA) weresynthesized from poly(ethylene glycol) or poly(ethylene glycol) diamine(Mw 3,400, Shearwater Polymers) as described in Elbert D L and Hubbell JA. “Conjugate addition reactions combined with free-radical crosslinkingfor the design of materials for tissue engineering,” Biomacromolecules2001;2:430441, which is incorporated by reference in its entirety.Gelatin from bovine skin (Types B and A, gel strength approx. 225bloom), Dulbecco's phosphate buffered saline (PBS), cystein, bovinetesticular hyaluronidase (HAse, 330 U/mg), bacterial collagenase fromClostriditim histolyticum (EC 3.4.24.3, 388 U/mg were obtained fromSigma Chemical Co. (St. Louis, Mo.). 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide (MTT) were obtained from Sigma Chemical Co.(St. Louis, Mo.). 5,5′-Dithio-bis(2-nitrobenzoic acid) (DINB) waspurchased from Acros (Houston, Tex.).

Analytical Instrumentation. Proton NMR spectral data were obtained usinga Varian INOVA 400 at 400 MHz. UV-Vis spectral data were obtained usinga Hewlett Packard 8453 UV-visible spectrophotometer (Palo Alto, Calif.).Gel permeation chromatography (GPC) analysis was performed using thefollowing system: Waters 515 HPLC pump, Waters 410 differentialrefractometer, Waters™ 486 tunable absorbance detector, Ultrahydrogel250 or 1000 columns (7.8 mm i.d.×130 cm) (Milford, Mass.) The eluent was200 mM phosphate buffer (pH 6.5)/MeOH=80:20 (v/v) and the flow rate was0.3 or 0.5 mL/min. The system was calibrated with standard HA samplesprovided by Dr. U. Wik (Phanmacia, Uppsala, Sweden). Fluorescence imagesof viable cells were recorded using a Nikon Eclipse TE300 withepi-fluorescence capabilities. Cell proliferation was determined using abiochemical assay (Cell-Titer 96 Proliferation Kit, Promega, Madison,Wis.), MTT assay, or MTS assay at 550 nm, which was recorded on an OPTIMx microplate reader (Molecular Devices, Sunnyvale, Calif.).

Synthesis of thioacid dihydrazides. The oxidized forms of the requiredthiol crosslinkers 3,3′-dithiobis(propanoic hydrazide) (DTP) and4,4′-dithiobis(butanoic hydrazide) (DTB) were synthesized from theirdiacids as described previously for DTP in Vercruysse, K. P.; Marecak,D. M.; Marecek, J. F.; Prestwich, G. D.; Bioconjugate Chem. 1997, 8,686-694, which is incorporated by reference in its entirety. Thus, freedicarboxylic acids were converted into diesters by refluxing in ethanolwith acid catalysis. The diesters were hydrazinolyzed with hydrazinehydrate to form the corresponding dihydrazides. DTP (Vercruysse, K. P.;Marecak, D. M.; Marecek, J. F.; Prestwich, G. D. Bioconjugate Chem.1997, 8,686-694): yield, 92%; ¹H NMR (400 MHz, DMSO-d₆): δ 9.05 (s, 2 H,N—NH—C(O)), δ 4.21 (s, 4 H, NH₂—N—C(O)), δ 2.88 (t, 4 H, C(O)—C—CH₂—S),δ 2.40 (t, 4 H, N—C(O)—CH₂—C). DTB: yield, 52%; ¹H NMR (400 M ,DMSO-d₆): δ 8.95 (s, 2 H, N—NH—C(O)), δ 4.15 (s, 4 H, NH₂—N—C(O)), δ2.66 (t,4 H, C—C—CH₂—S), δ 2.10 (t, 4 H, C(O)—CH₂—C—C), δ 1.82 (p, 4 H,C(O)—C—CH₂—C—S); MS-EI, m/z 266.0 (M⁺, 1.44); 133.0 (SC₃H₆CON₂H₃ ⁺,46.78); 101.0 (C₃H₆CON₂H₃ ⁺, 100.0). HRMS for C₈H₁₈O₂S₂N₄. Found266.0864; Calcd. 266.0871.

Preparation of low molecular weight (LMW) HA by acid degradation. Highmolecular weight HA (1.5 MDa) (20 g) was dissolved in 2.0 L distilledwater, and the solution pH was adjusted to ca. 0.5 by the addition ofconcentrated HCl. The degradation was carried out at 37° C., 130 rpmstirring for 24 h. After that, the pH of the solution was adjusted to7.0 by the addition of 1.0 N NaOH before transfer to dialysis tubing(M_(w) cut-off 3,500) and dialyzed against water for four days. Thesolution was then centrifuged, and the supernatant was lyophilized togive 15 g LMW HA (M_(w) 246 kDa, Mn 120 kDa, polydispersity index 1.97).

II. Disulfide Crosslinked Hyaluronan Hydrogels via Oxidative Addition

Preparation of Thiolated HA.

Thiolated HA derivatives with different loadings were prepared followinga general protocol (FIG. 1). In a representative example, LMW HA (20 g,50 mmol) was dissolved in 2.0 L of water, 23.8 g of DTP or 26.6 g of DTB(100 mmol) was added while stirring. The pH of the reaction mixture wasadjusted to 4.75 by the addition of 1.0 N HCl. Next, 19.2 g of EDCI (100mmol) in solid form was added. The pH of the reaction mixture wasmaintained at 4.75 with aliquots of 1.0 N HCl. The reaction was stoppedby addition of 1.0 N NaOH, raising the pH of the reaction mixture to7.0. Then, 100 g DTF (ca. 650 mmol) in solid form was added and the pHof the solution was raised to 8.5 by addition of 1.0 N NaOH. Afterstirring for 24 h, the pH of the reaction mixture was adjusted to pH 3.5by the addition of 1.0 N HCl. The acidified solution was transferred todialysis tubing (M_(w) cut-off 3,500) and dialyzed exhaustively againstdilute HCl (pH 3.5, approximately 0.3 mM) containing 100 mM NaCl,followed by dialysis against dilute HCl, pH 3.5. The solution was thencentrifuged, and the supernatant was lyophilized. The purity ofthiolated HA (HA-DTPH and HA-DTBH) was measured by GPC and ¹H NMR, andthe degree of substitution (SD) was determined by ¹H NMR. The freethiols on the side chain of HA-DTPH and HA-DTBH were determined by amodified Ellman method (Butterworth, P. H. W.; Baum, H.; Porter, J. W.Arch. Biochem. Biophlys. 1967, 118, 716-723). SD (%) and thiol content(%) were defined as the number of DTP (or DTB) residues and thiols per100 disaccharide units, respectively. Representative results: HA-DTBH(M_(w) 165 kDa, M, 63 kDa, polydispersity index 2.62, SD 72%) andHA-DTPH (M_(w) 136 kDa, M_(n) 61 kDa, polydispersity index 2.23, SD58%). The structures of HA-DTPH and HA-DTBH were confirmed by ¹H NMRspectroscopy in D₂O.

SD was mainly controlled by the molar ratios of HA/DTP/EDC and reactiontime (Table 1). By selecting suitable reaction parameters, the degree ofsubstitution can be controlled over a wide range (28% to 67%) (Table 1).Similar results were also observed in case of DTB-modified HA. TABLE 1Optimization of DTP modification of HA. molar ratio of reaction timeDegree of substitution HA:DTP:EDCI (min) (%) 1:2:2 5 28.8 1:2:2 10 41.71:2:2 30 49.2 1:2:2 60 54.4 1:2:2 120 66.8 1:1:2 10 38.9 1:4:2 10 42.51:2:1 10 31.1   1:2:0.5 10 26.8pK_(a) determination. The pK_(a) of thiols in HA-DTPH and HA-DTBH wasdetermined spectrophotometlically based on the UV absorption ofthiolates as proposed by Benesch and Benesch (Benesch, R.; Benesch, R.E. Proc. Nat. Acad. Sci. USA 1958, 44, 848-853). Solutions of HA-DTPHand HA-DTBH (ca. 5 mg) were dissolved in 100 mL 0.001 N HCl containing0.1 N NaCl (stable ionic strength). Freshly-prepared solutions wereimmediately measured in the UV region with a scan from 190 to 300 nm.

The pK_(a) values were determined spectrophotometrically based on the UVabsorption of thiolates (Benesch). With increasing pH, the absorption ofsolutions increased abruptly—especially at the pH near the pK_(a) ofthiols (FIG. 2 a). According to the procedure reported by Lutolf andco-workers (Lutolf, M. P.; Tirelli, N.; Ceiritelli, S.; Cavalli, L.;Hubbell, J. A. Bioconjugate Chem. 2001, 12, 1051-1056) the interceptwith the abscissa in a graphical representation oflog[(A_(max)−A_(i))/A_(i)] vs. pH yielded the pK, value. There was goodlinear approximation of the five central points both for HA-DTPH andHA-DTBH, giving a value of 8.87 for HA-DTPH and 9.01 for HA-DTBH. ThepK_(a) of HA-DTPH was slightly lower than that of HA-DTBH because thethiol in HA-DTPH was more easily activated by the proximity of the amidegroup (FIG. 2 b).

Compared to HA-DTBH, the lower pK_(a) of thiols in HA-DTPH suggestedincreased reactivity and increased capability to regenerate thedisulfide structure under the same conditions. A qualitative procedurewas used to evaluate the reformation of disulfide. When HA-DTPH andHA-DTBH solutions were in contact with air, the viscosity increased anda gel formed due to the oxidation of thiols to disulfide by O₂. Athigher pH, both HA-DTPH and HA-DTBH solutions more easily formed gelsbecause thiols were converted to more reactive thiolates at higher pH(Table 2). For instance, with 3.0% HA-DTPH solution (SD 58%), thesolution at pH 7.0, 8.0, and 9.0 gelled within 15 min, while at pH 5.0and 6.0 no obvious increase in viscosity of solution was observed after30 min (Table 2). The thiols of HA-DTBH were less reactive, and thus thegelation of HA-DTBH solution (3.0% w/v, SD 72%) was sluggish (Table 2).TABLE 2 The air-induced gelation of HA-DTPH and HA-DTBH solution (3.0%w/v) determined by a test tube inversion method. HA-DTPH HA-DTBH pH 15min 30 min 15 min 30 min 5.0 S S S S 6.0 S S S S 7.0 G G VS VS 8.0 G GVS G 9.0 G G VS GS = solution;G = gel;VS = highly viscous solutionGelation of thiolated HA solutions. The solution (flow)-gel (no flow)transition was determined by a flow test utilizing a test tube invertingmethod reported by Jeong et al. (Jeong, B.; Bae, Y. H.; Kim, S. W.Macromol. 1999, 32, 7064-7069). HA-DTBH and HA-DTPH were dissolved inPBS to give 3.0% (w/v) solutions under N₂ protection. The solution pHwas adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0 by 1.0 N NaOH.Freshly-prepared solutions (1.0 mL) with different pH were immediatelyinjected into glass tubes (0.8 cm in diameter, 7.5 cm in length). Afterexposure to air at room temperature for 15 or 30 min, the test tube wasinverted. If no fluidity was visually observed in 1 min, we concludedthat that a gel had formed.Preparation of disulfide-crosslinked HA films. HA-DTBH and HA-DTPH weredissolved in PBS to give 3.0% (w/v) solutions and the solution pH wasadjusted to 7.4 by the addition of 1.0 N NaOH. For drug-loaded gels,0.15% (w/v) blue dextran (M_(w) 200,000) was included as a model drug.Next, 25 mL of the solution was poured into a 9-cm petri dish andallowed to dry at room temperature. After ca. three days, a film ready.As required, the film was further oxidized by immersion in 0.3% H₂O₂ for1 h. The film was then rinsed with distilled water, cut into 6-mmdiameter discs, and dried at room temperature for one day and then at 1mm Hg for one week, to give films with 0.1 mm thickness.Swelling determination. Discs of HA-DTBH and HA-DTPH film (6 mm indiameter) were weighed (W₀), immersed in glass vials containing 10 mLPBS (pH 7.4), and placed in a shaking incubator at 37° C., at 300 rpm.At predetermined time intervals, the wet films were weighed (W₁)immediately after the removal of the surface water by blotting betweentwo pieces of filter paper. Swelling ratio (R) was defined as W_(t)/W₀.

The swelling of HA-DTPH and HA-DTBH films in PBS was in accordance withthe disulfide content in the films as shown in FIG. 3. The air oxidizedfilms swelled significantly because of low degree of crosslinking, witha swelling ratio at 5.5 h of 16.2 for HA-DTBH film and 9.5 for HA-DTPH.These ratios are similar to PEG-dialdehyde crosslinked HA adipicdihydrazide hydrogels used for drug release (Luo, Y., Kirker, K. R.;Prestwich, G. D. J. Controlled Rel. 2000, 69, 169-184) and wound repair(Kirker, K. R.; Luo, Y.; Nielson, J. H.; J.Shelby; Prestwich, G. D.Biomaterials 2002, 23, 3661-3671). After H₂O₂ oxidation, the swellingratio decreased to 3.5 for the HA-DTBH film and 2.9 for HA-DTPH film.

Disuifide content determination. Discs of HA-DTBH and HA-DTPH film weredegraded by acid hydrolysis (0.1 N HCl). The total sulfur content(disulfide plus thiol) was measured using 2-nitro-5-thiosulphobenzoate(NTSB) according to Thannhauser et al. (Thannhauser, T. W.; Konishi, Y.;Scheraga, H. A. Meth. Enzymol. 1987, 143, 115-119). In addition, thefree thiol content was measured by the Ellman method (Ellman, G. L.Arch. Biochem. Biophys. 1958, 74, 443-450). Disulfide content wascalculated from the difference between total sulfur content and freethiol content.

FIG. 4 shows the oxidation with dilute H₂O₂ increased the number ofdisulfide linkages. For example, the disulfide content in HA-DTPH filmincreased from 0.175 to 0.212 mmol/g after the oxidation of H₂O₂. In thecase of HA-DTBH film, fewer disulfide linkages were formed due to airoxidation because the thiol was less reactive (the value was 0.125mmol/g); however, this could be increased significantly to 0.25 mmol/gby oxidation with H₂O₂. However, following the H₂O₂ oxidizing procedure,no additional thiol groups are detected within both the HA-DTPH and theHA-DTBH films, and only ca. 30 to 40% of the available thiols formeddisulfide bonds. This suggests that H₂O₂ oxidation of thiol groups notonly created new disulfide bridges, but led to the production ofS-oxidized sulfenic or sulfonic acids that would not be detected usingNTSB and DTNB (Capozzi, G.; Modena, G. In The Chemistry of the ThiolGroup Part II; Patai, S., Ed.; Wiley: New York 1974; pp 785-839).

Blue dextran release studies. Drug-loaded 6-mm discs of HA-DTBH andHA-DTPH film were immersed in glass bottles containing 2 mL releasemedia, and placed in an incubator at 37° C., at 300 rpm. Atpredetermined time intervals, 1 mL supernatant solution was removed, andthe blue dextran content was determined by UV-vis absorption at 625 nm.Then, 1 mL blank release media was added back to maintain constantvolume. Release media: PBS containing 0, 10 and 50 mM DTT (the media pHwas adjusted to pH 7.4 by adding 1.0 N NaOH) or PBS containinghyalutonidase (HAse, 100 U/mL).

To further confirm the HA-DTPH and HA-DTBH films were crosslinked byreversible disulfide linkages, the hydrogel films were incubated in PBSthat contained different concentrations of DTT at pH 7.4 (data notshown). Even with DTT concentration as low as 10 mM, films generatedfrom both air and H₂O₂ oxidation swelled significantly and dissolvedgradually due to reduction of disulfide by DTF. As the gels dissolved, amodel drug (blue dextran M_(w) 200,000) that had been non-covalendyentrapped in the hydrogel films was released. Thus, within 100 min bothHA-DTPH films that had been further oxidized with H₂O₂ were dissolvedand consequently the blue dextran model drug was released completely inthe presence of DTT concentration of 10 mM and 50 mM. The release ofblue dextran occurring in the absence of DTT (FIG. 5) was negligible.

Furthermore, the enzyme HAse also accelerated the release of model drug(blue dextran) from films. For example, in 48 h the release percentageof blue dextran from air-oxidized HA-DTPH film in PBS at 37° C. at 300rpm was less than 7%, while under the same conditions in PBS with 100U/mL Hase, 30% of the blue dextran was released with concomitant partialdegradation of the film. After 48 h, approximately 36% of the film hadbeen lost due to enzymatic digestion, as determined gravimetrically.

In situ cell encapsulation. HA-DTPH (M_(w) 136 kDa, M_(n)=61 kDa,polydispersity index 2.23, SD=58%) was dissolved in DMEM/F-12 medium(GIBCO, Rockville, Md.) to give a 3.0% (w/v) solution under N₂protection, and the solution pH was adjusted to 7.4 by adding 1.0 NNaOH. Then the solution was sterilized under UV light for 25 min underN₂ protection. Murine fibroblasts (L-929, ATCC, Manassas, Va.) werecultured in a triple flask (Fisher, Springfield, N.J.) until 90%confluence, and then trypsinized and mixed with HA-DTPH solution to afinal concentration of 2×10⁶/mL. Next, 0.5 mL of the HA-DTPH solutionwas added into each well of a 12-well plate. The cell-loaded plates wereincubated (37° C., 5% CO₂, 4 h) until a solid hydrogel formed, and then2 mL of DMEM/F-12 medium with 10% of newborn calf serum (GIBCO,Rockville, Md.) was added into each well. The plates were transferred toan incubator (37° C., 5% CO₂, three days) without a change of medium.

Cell viability and proliferation. The viability of murine p929fibroblasts in the hydrogel was determined by a double-stainingprocedure using fluorescein diactate (F-DA) and propidium iodide (PI)(Kortemme, T.; Creighton, T. E. J. Mol. Biol. 1995, 253, 799-812). F-DA(Molecular Probes, Eugene, Oreg.), a non-fluoresent fluoresceinderivative, diffuses through the membrane of living cells and ishydrolyzed by intracellular esterase to produce a green fluorescence. PI(Sigma Chemical Co., St. Louis, Mo.), which is excluded by intact cellmembranes, but was able to diffuse across a damaged cell membrane, bindsto nucleic acids to produce a bright red fluorescence. Briefly, a 5mg/ml solution of F-DA in acetone was diluted to 20 μg/ml in PBS thatcontained 0.2 μg/ml PI. After 1 and 3 days culture with encapsulatedcells in vitro, the hydrogels were rinsed twice with PBS, immersed inthe diluted F-DA/PI solution for 10 min at room temperature and thenwashed with PBS for 5 min. Then, live and dead cells were observed on aNikon TS 100 microscope (Nikon, Melville, N.Y.) with Triple(DAPI/FITC/CY3) filter.

After different culture times, the number of viable cells in eachhydrogel was determined using a biochemical assay (Cell-Titer 96Proliferation Kit, Promega, Madison, Wis.) as previously described(Lutolf, M. P.; Tirelli, N.; Cerritelli, S.; Cavalli, L.; Hubbell, J. A.Bioconjugate Chem. 2001, 12, 1051-1056). In this method, a tetrazoliumsalt (MTS) is reduced by the mitochondria of living cells into a coloredformazan product whose presence can be detected spectrophotometrically.

The hydrogels in 12-well plates were rinsed twice with PBS buffer, then900 μl of DMEM/F-12 medium with 5% of newborn calf serum and 180 μL ofCell Titer 96 Proliferation Kit solution were added into each well.After 2 h of incubation with gentle shaking (37° C., 5% CO₂), a 125-μLaliquot of each of the solutions was transferred individually into a96-well plate and read at 550 nm with a OPTI Max microplate reader(Molecular Devices). The absorbance reading was converted into a cellnumber based on standard curves generated from the assay of knownnumbers of cells. Data sets were compared using two-tailed, unpairedt-tests. P-values less than 0.05 were considered to be significant.

The rapid gelation of HA-DTPH solution under physiological conditionsexhibits potential utility for many biomedical applications, e.g., woundhealing, defect filling, prevention of post-surgical adhesions, and cellencapsulation for tissue repair. Murine fibroblasts were entrappedwithin a crosslinking HA-DTPH hydrogel, and the encapsulated cells wereexamined after 24 h and 96 h of culture. Viable cells, indicated bygreen fluorescence upon F-DA staining, were evident after 96 h ofculture. Fewer than 5% dead cells were observed as red fluorescence fromPI staining (data not shown). Unlike two-dimensional culture in flasks,the fibroblasts in the hydrogel maintained a round shape. In addition,clumps of cells, as well as individual cells, were observed in hydrogel.

After in vitro culture for 1, 2, and 3 days, the number of viable cellsresiding in the hydrogel were determined by MTS assay (Cell-Titer 96Proliferation Kit, Promega, Madison, Wis.). The results indicated thatcells proliferated in hydrogel after culture of 2 and 3 days, and thecell number increased ca. 15% at day 3, which was significant withp<0.05 (FIG. 6).

III. Disulfide Crosslinked Hyaluronan-Gelatin Hydrogels

Synthesis of thiolated HA and gelatin. Low molecular weight (LMW) HA (Mw246 kDa, Mn 120 kDa, polydispersity index 1.97) was prepared bydegradation of high molecular weight HA (1.5 MDa) in dilute HCl (pH ca.0.5) for 24 h at 37° C. 150 rpm. Thiolated HA and gelatin weresynthesized separately following a general protocol as previouslydescribed for HA and chondroitin sulfate (CS) modification (FIG. 7).Thus, 20 g of LMW HA (50 mmol) or 20 g of gelatin was dissolved in 2.0 Lof water, and then DTP (11.9 g, 50 mmol for HA; 20 g for gelatin) wasadded while stirring. The pH of the reaction mixture was adjusted to4.75 by the addition of 1.0 N HCl. Next EDCI (4.8 g, 25 mmol for HA; 10g for gelatin) was added in solid form. The pH of each reaction mixturewas maintained at 4.75 by the addition of aliquots of 1.0 N HCl. Thereaction was stopped by addition of 1.0 N NaOH to increase the pH to7.0. Then, 100 g of DTT (ca. 650 mmol) was added in solid form and thepH of the solution was further increased to 8.5 by addition of 1.0 NNaOH. After stirring for 24 h at ambient temperature, the pH of thereaction mixture was adjusted to pH 3.5 by the addition of 1.0 N HCl.The acidified solution was transferred to dialysis tubing (MWCO 3,500)and dialyzed exhaustively against ca. 0.3 mM HCl solution (pH 3.5)containing 100 mM NaCl, followed by dialysis against 0.3 mM HCl (withoutsalt) at pH 3.5. The solution was then clarified by centrifugation, andthe supernatant was lyophilized. The purity of thiolated HA (HA-DTPH)and thiolated gelatin (gelatin-DTPH) were measured by GPC and ¹H NMR,and the degree of substitution (SD) and the free thiols on the sidechain of HA-DTPH and gelatin-DTPH were determined by ¹H NMR and by amodified Ellman method (Butterworth P R W, Baum H, and Porter J W. Amodification of the Ellman procedure for the estimation of proteinsulfhydryl groups. Arch Biochem Biophys 1967;1 18:716-723).

pKa determination. The pKa values for the thiols in HA-DTPH andgelatin-DTPH were determined spectrophotometrically based on the UVabsorption of thiolates (Benesch R and Benesch RE. Thiolation ofprotein. Proc Nat Acad Sci USA 1958;44:848-853; Lutolf M P, Tirelli N,Cerritelli S, Cavalli L, and Hubbell J A. Systematic modulation ofMichael-type reactivity of thiols through the use of charged aminoacids. Bioconjugate Chem 2001; 12:1051-1056). Solutions of HA-DTPH andgelatin-DTPH (ca. 5 mg each) were dissolved in 100 ml of 0.001 N HClcontaining 0.1 N NaCl (stable ionic strength). UV scans from 190-300 nmwere recorded for freshly-prepared solutions,

Turbidimetric titration. The electrostatic interactions of HA-DTPH andgelatin-DTPH were investigated by turbidimetric titration (Shu X Z, ZhuK J, and Song W. Novel pH-sensitive citrate crosslinked chitosan filmfor drug crontrolled release. Int J Pharm 2001;212: 19-28; Park J M,Muhoberac B B, Dubin P L, and Xia J. Effects of protein chargeheterogeneity in protein-polyelectrolyte complexatiom Macromolecules1992;25:290-295). A solution of 1.0 mg/ml of either HA-DTPH or LMW HAand 1.0 mg/ml of either gelatin-DTPH or unmodified gelatin was preparedat pH 1.5, and aliquots of a stock NaCl solution were added to adjustthe ionic strength. Titrant (0.01-0.2 N NaOH) was delivered using amicroburette into the solution with gentle stirring at 30 plus/minus0.5° C., and the pH was monitored by a digital pH meter with a precisionof plus/minus 0.01. Changes in turbidity were monitored at 420 nm withan WV-vis spectrophotometer and reported as (100-T)%, which is linearlyproportional to the true turbidity measurements when T>0.9. The timeinterval between turbidity measurements was ca. 4 min.

Next, HA-DTPH and gelatin-DTPH were dissolved in 0.02 M PBS (pH 6.5) togive 3.0% (w/v) solutions. The pH of each solution was adjusted to 7.4by the addition of 1.0 N NaOH, and then the solutions were mixedaccording to volume ratio of HA-DTPH:gelatin-DTPH of 100:0, 80:20,60:40, 40:60, 20:80, and 0:100. At different times, the transmittance ofthe solutions was monitored at 550 nm.

Turbidimetric titration revealed that there were ionic interactionbetween LMW HA and gelatin, with the formation of a polyelectrolytecomplex in the pH range 2.3-5.0, where HA was negatively charged whilegelatin (Type B, pI=4.9) was positively charged (data not shown). Thisphenomenon was evaluated for the thiolated derivatives of HA andgelatin, which still have numerous unmodified carboxylates (1.58 mmol/gfor HA-DTPH, 0.65 mmol/g for gelatin-DTPH) and amine groups (0.35 mmol/gfor gelatin-DTPH). Turbidometric titration indicated that similarelectrostatic interactions occurred in the mixed solutions of HA-DTPHand getatin-DTPH, but over a broader pH region due to the shift tohigher pI for gelatin-DTPH resulting from conversion of >40% of thecarboxylates to thiols.

HA-DTPH and gelatin-DTPH were dissolved in 0.02 M PBS, and the pH wasadjusted to 7.4 to give clear solutions. When mixed, solutionscontaining various ratios of HA-DTPH and gelatin-DTPH becametranslucent, and phase separation occurred immediately due to theirelectrostatic interactions (FIG. 8). This effect precluded fabricationof homogeneous, transparent hydrogel films from blends of HA-DTPH andgelatin-DTPH. To overcome the formation of polyelectrolyte complexes,the ionic strength of the solutions was increased to mask theelectrostatic binding. Indeed, turbidimetric titration revealed thatthis binding was completely prevented by 3.0% (w/v) NaCl (data notshown). However, this high concentration of salt disturbed the filmformation and resulted in an unacceptably brittle film. Therefore, 1.0%(w/v) NaCl was added into HA-DTPH and gelatin-DTPH solution to permitthe formation of clear solutions at ratios of HA-DTPH to gelatin-DTPH of80:20, 60:40, 40:60, and 20:80. No phase separation occurred in 2 h(FIG. 8), although after 24 h, these blended solutions also becameopaque, indicating the persistence of electrostatic interactions betweenthese two macromonomers.

Preparation of HA-gelatin hydrogel films crosslinked by disulfide bond.HA-DTPH and gelatin-DTPH (3.0 g each ) were separately dissolved in 100ml of 20 mM PBS buffer (pH 6.5) containing 1.0% (w/v) NaCl, and then thepH of each solution was adjusted to 7.4 by the addition of 1.0 N NaOH.Then, HA-DTPH and gelatin-DTPH solutions were combined in volume ratiosof 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100, and thoroughly mixed bygentle vortexing. The mixed solutions (30 ml) were poured into 9-cmpetri-dishes and allowed to crosslink in air and to dry at roomtemperature. After 3 days, air-crosslinked films were obtained and cutinto 6, 8, or 1.6-mm diameter discs. The film discs were then furtheroxidized by immersion in 0.1% H₂O₂ for 1 h. The film discs were thenrinsed with distilled water and dried at ambient pressure andtemperature for one day, and then at 1 mm Hg for one week.

Based on the above results, 1.0% NaCl was used to shield theelectrostatic interaction between HA-DTPH and gelatin-DTPH during filmformation and crosslinking. The blended hydrogel films were obtained bypouring 30 ml of mixed HA-DTPH-gelatin-DTPH solutions containing 1.0%NaCl (w/v) into 9-cm petri-dishes. Air oxidation and drying at roomtemperature produced disulfide-crossslinked films. Crosslinking densityin these films was increased by further oxidation with 0.1% (w/v) H₂O₂;films were then rinsed and dried in vacuo.

The disulfide content of the HA-gelatin hydrogel films was determined byNTSB after exhaustive acidic hydrolysis (FIG. 9). In agreement withprevious results (Nicolas F L and Gagnieu C H. Denatured thiolatedcollagen II. Crosslinking by oxidation. Biomaterials 1997;18:815-821),only 25-50% of the thiols were oxidized to disulfides. Since no freethiols were detectedby DTNB (Ellman G L. A calorimetric method fordetermining low concentrations of mercaptans. Arch Biochem Biophys1958;74:443-450), this indicated that the other thiols were oxidized byH₂O₂ to S-oxidized sulfinic, sulfenic, or sulfonic acids that would notbe detected using NTSB and DTNB (Capozzi G and Modena G. Oxidation ofthiol. In: Patai S, editor. The Chemistry of the Thiol Group Part II.New York: Wiley, 1974, p. 785-839). However, in contrast with thethiolated HA alone, a high proportion of disulfide-crosslinking wasobserved in the gelatin-DTPH film, despite the lower acidity of thethiols. Clearly, additional factors, such as the more flexibleconformation of the modified gelatin and more mobile, longerthiol-containing side chain could facilitate disulfide formation. Forthe more rigid linear polysaccharide HA, ca. 25% of the theoreticaldisulfide bonds were formed in the HA-DTPH hydrogel film; however, over50% of the theoretical disulfide bonds were formed in gelatin-DTPHhydrogel film. Thus, even though the thiol concentration in HA-DTPH(0.768 mmol/g) is higher than for gelatin-DTPH (0.512 mmol/g), asignificantly higher disulfide content was found for the gelatin-DTPHfilm (0.123 mmol/g) relative to the HA-DTPH film (0.100 mmol) (p<0.02).Electrostatic attraction between HA-DTPH and gelatin-DTPH alsofacilitated disulfide formation; blended films had more disulfide bondsthan the HA-DTPH film (p<0.01, except for HA-DTPH:gelatin-DTPH of80:20). The disulfide density of the films with ratio ofHA-DTPH:gelatin-DTPH of 40:60 (0.136 mmol/g) and 20:80 (0.145 mmol/g)even higher than that in gelatin-DTPH film (0.123 mmol/g) (p<0.01).

Swelling determination. Film discs with diameter of 6 mm were weighed(Wd), immersed in glass vials containing 10 ml PBS (pH 7.4), and placedin an incubator at 37° C., 300 rpm. At predetermined time intervals, thewet films were weighed 15 (Wt) immediately after the removal of thesurface water by blotting briefly between two pieces of filter paper.The swelling ratio (R) was defined as Wt/Wd.

The equilibrium swelling ratio of the hydrogel films in PBS is shown inFIG. 10. With increasing percentages of gelatin-DTPH, the swelling ratiodecreased from 3.27 to 2.33. This ratio is determined only by thecrosslinking density, but is also related to the bulk properties of thefilms.

One of the disadvantages for many HA and gelatin-based biomaterials israpid degradation in vivo. Therefore, the optimization of the mechanicalproperties and rate of degradation by co-crosslinking the HA and gelatinwas sought. This strategy proved effective. Preliminary evidence showedthat disulfide-crosslinked HA-DTPH hydrogels would degrade slowly, bothin vitro and in vivo, and that the degradation rate could be controlledby altering the disulfide-crosslinking density. Thus, approximately 30%weight loss of HA-DTPH hydrogel film was found after 42 days ofimplantation in vivo in mice (data not shown). With a very highconcentration of HAse (300 U/ml) in vitro, only ca. 8% weight loss ofHA-DTPH hydrogel film was observed in 48 h (data not shown). On theother hand with the same concentration of enzyme (collagenase 300 U/ml),in 48 h, ca. 62% of gelatin-DTPH hydrogel film was digested (data notshown). The rapid degradation of gelatin-based biomaterials could limitusage in many biomedical applications (Nicolas F L and Gagnieu C H.Denatured thiolated collagen II. Crosslinking by oxidation. Biomaterials1997;18:815-821).

Disulfide content determination. Film discs with diameter of 6 mm weredegraded by acid hydrolysis (0.1 N HCl, 37° C., 150 rpm for 10 days).The total sulfur content (S−S+SH) was measured using2-nitro-5thiosulfobenzoate (NTSB) (Thannhauser T W, Konishi Y, andScheraga H A. Analysis for disulfide bonds in peptides and proteins.Methods In Enzymology 1987;143:115-119), and the free thiol content wasmeasured by the Ellman method (Ellman G L. A colorimetric method fordetermining low concentrations of mercaptans. Arch Biochem Biophys1958;74:443-450). Disulfide content, equivalent to crosslinking density,was calculated as the difference between total sulfur content and freethiol content.

In vitro degradation of HA-gelatin hydrogel film. The degradation ofdisulfide-crosslinked HA-gelatin films was performed using collagenaseand HAse. Film discs with diameter of 8 mm were incubated in a glassbottle containing 3 ml medium with 300 U/ml collagenase or HAse, andplaced in an incubator at 37° C., 150 rpm. The medium was changed everytwo days. At predetermined intervals, the films were washed five timeswith distilled water and dried under vacuum. The buffer used forcollagenase was 100 mM Tris-HCl buffer (pH 7.4) containing 5 mM CaCl₂and 0.05 mg/ml sodium azide (Choi Y S, Hong S R, Lee Y M, Song K W, ParkM H, and Nam Y S. Studies on gelatin-containing artifical skin:II.preparation and characterization of crosslinked gelatin-hyaluronatesponge. J Biomed Mater Res (Appl Biomater) 1999;48:631-639). HAsedigestions were performed in 30 mM citric acid, 150 mM Na₂HPO₄, 150 mMNaCl (pH 6.3) (Bulpitt P and Aeschlimann D. New strategy for chemicalmodification of hyaluronic acid: Preparation of functionalizedderivatives and their use in the formation of novel biocompatiblehydrogels. J Biomed Mater Res 1999;47:152-169). For simultaneousdigestion with collagenase and HAse, the buffer used was 100 mM.Tris-HCl buffer (pH 7.4) containing 5 mM CaCl₂, 150 mM NaCl, and 0.05mg/ml sodium azide. The weight loss fraction was determined as(1-Wt/W0), where Wt is the weight of dried film at time t, and W0 is theoriginal weight of dried film.

The blending of HA-DTPH with the gelatin-DTPH in films significantlyslowed digestion by collagenase. Thus, with 300 U/ml collagenase,HA-gelatin films (80% or 60% gelatin), the weight loss in two days wasonly 15% and 2%, respectively (FIGS. 11 a and 11 b). These films werealso more resistant to the digestion by HAse than HA-DTPH films, withless than 5% weight loss in 300 U/ml HAse for two days (FIGS. 11 a andb). When both HAse and collagenase were present, degradation ofHA-gelatin film was accelerated. For instance, the weight loss after 7days for an HA-gelatin (60% gelatin) film was 18% with 300 U/mlcollagenase and 5% with HAse; with both enzymes combined, weight losswas as high as 50% (FIG 11 b).

Cell growth on the surface of hydrogel films. The growth of murineBalb/c 3T3 fibroblasts (ATCC) on disulfide-crosslinked HA/gelatinhydrogel film was evaluated. The cells were cultivated in Modified EagleMedium (DMEM, GIBCO) supplemented with 10% newborn calf serum (GIBCO),Pen-Strep, L-glutamine and sodium bicarbonate. The fibroblasts weretrypsinized in the logarithmic growth state and evenly seeded onto thehydrogel surfaces at ca. 5,000 or at 25,000 cells/cm².

Cell viability. An in situ fluorescence viability assay with fluoresceindiacetate (F-DA, Molecular Probes, Eugene, Oreg.) was performed toassess the cell viability on the hydrogel surface. A 5 mg/ml solution ofF-DA in acetone was prepared and diluted to 0.02 mg/ml in PBS. After 24h of in vitro culture in an incubator with 5% CO₂ at 37° C. (25,000cells/cm² were initially seeded), the hydrogel films were rinsed withPBS twice to remove the unattached cells, and then immersed in thediluted F-DA solution for 3 min at room temperature and then washed inPBS for 5 min. Fluorescence in the live cells was observed using a NikonTS 100 microscope (Nikon, Melville, N.Y.) with DAPI filter, andphotomicrographs of the cell attachment and spreading were recorded.

Balb/c 3T3 fibroblasts were seeded on the surface of the HA-gelatinfilms of different compositions and cultured in vitro for 24 h, and thenthe live cells were stained with F-DA to give green fluorescence. Amorphological study revealed that only a very small number of cells withspherical shape were attached to the surface of HA-DTPH hydrogel filmthat lacked a protein component (FIG. 12 a). Addition of gelatin-DTPHsignificantly improved the cell attachment (FIG. 12 b-12 d), even at 20%(w/v). At gelatin concentrations of 40% and higher, the majority ofcells adopted a spindle-shaped morphology and spread uniformly on thehydrogel surface (FIG. 12 b-12 d).

Cell proliferation. The surfaces of 2-cm² film discs were seeded with5,000 cells/cm². After 24 and 72 h of incubation without changing thecell culture media, the cell numbers were evaluated by the metabolicreduction of MTT to a colored formazan dye by viable cells. Thus,sterile aliquots of a 5 mg/ml stock solution of MTT in PBS were added ata ratio of 60 1 per 500 1 of medium to each film disc (2 cm²) andincubated for 4 h at 37° C. Then, the medium was discarded, and eachfilm disc was incubated in 1.0 ml DMSO to lyse the cells and dissolvethe dyes. Cell-fiee film discs were used as blanks. Next, 200 μl of eachDMSO solution was transferred into a 96-well plate anid the absorptionwas recorded at 550 nm on an OPTI Max Microplate Reader.

Using cell culture-grade polystyrene as control, the proliferation ofBalb/c 3T3 fibroblasts on the hydrogel surface was evaluated. The cellswere initially seeded at a density of 5,000 cells/cm², and cultured invitro for one day and three days. and then the cell number wasdetermined by MTT assay. The cell number in cell culture polystyreneafter one day culture was defined as 1.0 and the relative cell densitywas calculated. FIG. 13 shows that while cells on the HA-DTPH hydrogelsurface failed to proliferate, increasing percentages of gelatin in thefilms result in accelerated cell proliferation. After three days culturein vitro, the cell number on the hydrogel surface with a gelatinpercentage greater than 60% (w/v), was more than 50% of the polystyrenecontrol, while the cell number on gelatin-DTPH hydrogel surface was 85%of the control.

Statistical analysis. Data sets were compared using two-tailed, unpairedt-tests; values of p<0.05 were considered to be significant

IV. Preparation of Hydrogels via Michael Addition

Synthesis of thiolated HA and thiolated gelatin. Low molecular weight(LMW) HA (M_(w) 246 kDa, M_(n) 120 kDa, polydispersity index 1.97) wasused after the degradation of high molecular weight HA (1.5 MDa) indilute HCl (pH 0.5) for 24 h at 37° C. 150 rpm. Thiolated HA (HA-DTPHand HA-DTBH) and thiolated gelatin (gelatin-DTPH and gelatin-DTBH) weresynthesized as described above. The degree of substitution (SD), i.e.,the fraction of carboxylates modified, was calculated from theintegrated ¹H-NMR spectrum.

Synthesis of Homobifunctional PEG Electrophiles

PEG-diacrylate (PEGDA), PEG-dimethacrylate (PEGDM), PEG-diacrylamide(PEGDAA) and PEG-imethacrylainide (PEGDMA) were synthesized from PEG (Mw3400 KDa, Aldrich) or PEG-diamine (M_(w) 3400, Shearwater Polymers) asdescribed with minor modifications. Briefly: PEG (or PEG-diamine)molecular weight 3400 (10 g, 5.88 mmol of functional group) wasazeotropically distilled with 400 ml of toluene under argon, removingca. 100 ml of toluene. The anhydrous solution was cooled at roomtemperature under argon and then cooled in an ice bath. Anhydrousdichloromethane (Aldrich) (ca. 50 ml) was added until the solutionbecome clear. Triethylamine (1.23 ml, 8.82 mmol, Aldrich) was addeddropwise with stirring, followed by the dropwise addition of 0.72 ml ofacryloyl chloride (8.82 mmol, Aldrich) or 0.85 ml of methacryloylchloride (8.82 mmol, Aldrich). The reaction was stirred in the dark,overnight under argon. The solutions were then filtered under vacuumuntil clear, and the product was precipitated in diethyl ether,collected by filtration and dried under vacuum. Next, 10 g of theproduct were dissolved in 10 ml of distilled water, adding 5 g of NaCl(the pH was adjusted to 6). The derivatives were then extracted 3 timeswith dichloromethane and precipitated in diethyl ether, and collected byfiltration and dried under vacuum. PEG diacrylate: yield 75%. ¹H-NMR(DCCl₃): 3.6 ppm (303.5 H, PEG), 4.3 ppm (t, 4H, —CH₂—CH₂—O—CO—CH═CH₂),5.8 ppm (dd, 2H, CH₂═CH—COO), 6.1 ppm, 6.4 ppm (dd, 4 H, CH₂═CH—COO—).Degree of substitution 95%. PEG dimethacrylate: yield 60%. ¹H-NMR(DCCl₃): 2.3 ppm (s, 6 H, CH₂═C(CH₃)—COO—), 3.6 ppm (303.5 H, PEG), 4.3ppm (t, 4H, —CH₂—CH2—O—CO—C(CH₃)═CH₂), 5.8 ppm, 6.1 ppm (d, 4 H,CH₂═C(CH₃)—COO—). Degree of substitution 91%. PEG diacrylamide: yield75%. ¹H-NMR (DCCl₃): 3.6 ppm (304.4 H, PEG), 5.6 ppm (dd, 2 H,CH₂═CH—CON—), 6.1 ppm and 6.3 ppm (dd, 4 H. CHCH—COO—). Degree ofsubstitution 100%. PEG dimethacrylamide: yield 71%. ¹H-NMR (DCCl₃): 2ppm (s, 6 H, CH₂═C(CH₃)—CON—), 3.6 ppm (304.4 H, PEG), 5.3 ppm, 5.8 ppm(d, 4 H, CH2═C(CH₃)—CON—). Degree of substitution 100%.

Conjugate addition. The relative reactivity of conjugate addition ofαβ-unsaturated esters and amides of poly(ethylene glycol) to thiols wasfirst evaluated using cysteine as a model. The conjugate addition ofcysteine to each of the four electrophilic species is shown in FIG. 15.Cysteine (2.5 mg) and PEG-diacrylate (PEGDA), PEG-dimethacrylate(PEDMA), PEG-diacrylamide (PEGDAA) or PEG-dimethacrylamide (PEGDMA) weredissolved in 5 ml of 0.1 N PBS, pH 7.4 (ratio of double bond/SH 2/1).Then the consuming of thiols was monitored by DTNB (Ellman) or NTSB(Thannhauser). Next, the conjugate addition of thiols with differentreactivity (i.e., different pKa values) was evaluated using the MW 375monofunctional PEG-acrylate as a model compound. HA-DTPH or HA-DTBH (10mg) was dissolved in 5 ml of 0.1 N PBS, pH 7.4, and then PEG-acrylatewas added (double bond:thiol=10:1). The consumption of free thiols wasmonitored using DTNB (Ellman, Thannhauser).

Hydrogel preparation. Thiolated HA and/or thiolated gelatin weredissolved in cell culture medium to give 1.25% (w/v) solution, and thepH was adjusted to 7.4. Two α,β-unsaturated ester and twoα,β-unsaturated amide derivatives of PEG were synthesized and used tocrosslink thiolated HA and gelatin (FIG. 14). Each of the four PEGderivatives (PEGDA, PEGDM, PEGDAA, and PEGDMA) was dissolved in PBS togive 4.5% (w/v) stock solution. Then, 1 ml of the stock reactive PEGsolution was added in one portion to 4 ml of the thiolated HA, thiolatedgelatin solution, or a blend of the two components, and mixed for 30seconds. Gel formation occurred within 10 min (PEGDA) to several days(PEGDMA), with the time dependent on the structure of the reactive PEGderivative. The conjugate addition of HA-DTPH to a low molecular weightPEG-monoacrylate was a slightly faster than between with the lessreactive HA-DTBH (FIG. 16).

Determination of Crosslinking Efficiency

Crosslinking was evaluated in detail for both thiolated HA deriviativeswith PEGDA as the homobifunctional crosslinker. After 1 h, a mixture ofeach thiolated HA (HA-DTPH and HA-DTBH) and PEGDA had completely gelled.The resulting hydrogels were then incubated in medium (pH 4.5 or 1.0) toquench the crosslinking addition, and the crosslinking efficiency wasdetermined by measuring the remaining free PEG electophile and theremaining free thiols and performing the calculations indicated below.

First, the quantity of free PEGDA in the hydrogel was determined by GPCwith monitoring of the eluent at 233 nm. Briefly, the hydrogel (0.1 ml)was ground into small particles and suspended in 2 ml of 0.1 M acetatebuffer (pH 4.5). After stirring for 4 h at room temperature, the amountof residual PEG derivatives was determined using a standard calibrationcurve. No free thiolated HA was detected by GPC at 210 nm.

Next, the free thiols in the hydrogel were determined using either theDTNB or NTSB assay. Briefly, a 0.05-ml fragment of hydrogel wassuspended in 0.5 ml of 0.1 N HCl solution. After 48 h at roomtemperature with agitation at 150 rpm, the hydrogel had dissociated.Next, 2.0 ml of either NTSB or DTNB reagent was added to each gel, andthe number of free thiols in the hydrogel was determinedspectrophotometrically at 412 nin. Thiolated HA solutions alone wereused as reference materials, and the disulfide formation during hydrogelpreparation (1 h) under nitrogen protection was negligible.

The extent of effective crosslinking (i.e., double-end anchorage),unreacted pendent double bond groups during the coupling reaction (i.e.,single-end anchorage) was calculated from the total PEGDA used (A), theunreacted PEGDA (B), the total thiols (C) and the free thiols inhydrogel (D). Single-end anchorage equals to the theoretical consumedthiols (2(A-B)) minus the actually consumed thiols (C-D). Subtraction ofsingle-end anchorage from the experimentally measured consumed thiols(C-D) reveals the extent of double-end anchorage. Table 3 shows thecrosslinking efficiencies, and Table 4 shows the crosslinking densities,equilibrium swelling ratios, and gelation times for the gels obtained bythe reaction of HA-DTPH and HA-DTBH. TABLE 3 Crosslinking efficiency ofPEGDA to HA-DTPH and HA-DTBH Crosslinking efficiency (%) Molar ratio ofPEGDA of thiols to Double-end Single-end double bonds anchorageanchorage Unreacted HA-DTPH:PEGDA 1:1 76.2 9.7 14.1 2:1 93.7 6.3 0 3:1100.0 0 0 HA-DTBH:PEGDA 1:1 48.3 19.3 32.4 2:1 60.0 12.7 27.3 3:1 73.88.3 17.9

TABLE 4 Crosslinking density, equilibrium swelling ratio (Q) andgelation time for gels prepared using PEGDA (Mw 3400) with HA-DTPH andHA-DTBH Molar ratio of thiols to Crosslinking Swelling Gelation doubledensity ratio time bonds (mmol/ml)* (Q) (min) HA- 1:1 8.1 39.41 ± 0.34 5 DTPH:PEGDA 2:1 5.0 46.15 ± 0.38  9 3:1 3.5 61.06 ± 0.89 19 HA- 1:15.1 58.14 ± 0.94 11 DTBH:PEGDA 2:1 3.2 69.33 ± 2.94 19 3:1 2.6 84.62 ±1.98 31*Crosslinking density was defined as the number of effectivecrosslinking sites in 1 ml of hydrogel.Swelling Determination

Hydrogels were placed in PBS buffer at 37° C. for 48 h and the mediumwas changed frequently. The swelling ratio (Q) was defined as a ratio ofthe weight of swollen gel to the weight of dry gel. The weight of thedry gels was determined by washing the hydrogel with distilled water 5times and then drying the gel under vacuum (1 mm Hg) at room temperaturefor 3 days.

The degradation of hydrogel. Hydrogel discs (0.5 ml) were prepared fromHA-DTPH and PEGDA as described above by crosslinking in the bottom of a6-mm diameter vial. Hyaluronidase (HAse) solutions (0, 50, 150 and 250U/ml) were prepared in 30 mM citric acid, 150 mM Na₂HPO₄, 150 mM NaCl(pH 6;3); 5 ml of enzyme solution was added to each vial containing thehydrogel, and vials were incubated at 37° C. with orbital agitation at150 rpm. The degradation of the gel was determined from the release ofglucuronic acid into the supernatant as measured by the carbazole assay(Bitter T and Muir H. A modified uronic acid carbozole reaction. Anal.Biochem. 1962;4:330-334). FIG. 17 shows the digestion of aHA-DTPH/-PEGDA hydrogel by HAse, showing that at lower concentrations ofenzyme the gel remains largely intact for several days in vitro.

In Vitro Cell Culture

Preparation of composites of T31 fibroblasts and HA-DTPH/PEG-diacrylatehydrogel. HA-DTPH solution (1.25%(w/v)) was prepared by dissolvinglyophilized HA-DTPH (SD=42%) in complete DMEM/F-12 medium, adjustedpH=7.4˜7.5 with 1.0N NaOH, and sterilized by filtration with 0.45 μmsyringe filter. Next, a 4.5% PEGDA solution was prepared by dissolvingPEGDA in PBS buffer and sterilized by filtration with 0.45 μm syringefilter. Then, T31 human pharyngeal fibroblasts that had been cultured intriple flasks (175 cm²) and trypsinized with 0.25% sterile trypsin in0.05% EDTA, were suspended in freshly prepared HA-DTPH solution atconcentration of 10⁶ cells/ml. To four volumes of the cell suspensionwas added one volume of the PEGDA stock solution, and the mixture wasvortexed gently. Next, 300 μl of the mixture of the fibroblast-seededHA-DTPH-PEGDA mixture was poured into each well of 12-well plate andgelation was allowed to occur (1 h). Finally, complete DMEM/F-12 mediumwas added into each well and the plate was incubated for at 37° C. in a5% CO₂ incubator. The medium was changed every three days withoutdamaging the gel. The seeded hydrogels were used to determine in vitrocell viability and proliferation and for transplantation in vivo intonude mice for fibrous tissue generation.

Cell viability and proliferation. Viability was determined withlive-dead staining methods at day 6 and day 28 of culture in vitro. Ateach time, four fibroblast-seeded HA-DTPH-PEGDA hydrogels were rinsedtwice with PBS buffer, stained for 3 min with fluorescein diacetate(F-DA, 0.02 mg/ml) and propidium iodide (PL 0.2 μg/ml) at roomtemperature, rinsed twice with PBS buffer, stored on ice, and observedusing a confocal microscope. The density of living cells in the gel wasdemonstrated by in situ fluorescence staining, and greatly increasedafter 28 days culture in vitro compared with that of 6 days. No deadcells were found as demonstrated by the absence of PI staining. FIG. 18shows the viability after 28 days of culture in vitro.

Cell proliferation was determined at day 0, 3, 6, 14, and 28. At eachtime, four fibroblast-seeded HA-DTPH-PEGDA hydrogels were transferredinto each well of a 12-well plate, and rinsed twice with PBS buffer.Next, 900 μL of DMEM/F-12 medium with 5% newborn calf serum and 180 μlof CellTiter 96 Proliferation Kit (Promega, Madison, Wis.) were addedinto each well of a 12-well plate. After 2 hr at 37° C. in a 5% CO₂incubator on an orbital shaker, 125 μl of the solution was transferredinto each of six wells of a 96-well plate. Absorbance (λ=550 nm) wasmeasured using an OPTI Max microplate reader (Molecular Devices,Sunnyvale, Calif.) and was converted into cell number based on astandard curve. The number of fibroblasts in the HA-DTPH-PEGDA hydrogelincreased almost tenfold after 28 days of culture ini vitro (FIG. 19).

Collagen Typing

At each time point (day 0, 3, 6, 14, and 28), four fibroblast-seededHA-DTPH-PEGDA hydrogels were minced with 22 gauze needles, digested in5% cyanogen bromide (CNBr, Sigma) in 70% formic acid (Sigma) for 8 h at35° C., diluted with same volume of distilled water, and lyophilizedovernight. The lyophilized samples were dissolved in PBS buffer and readwith Cary 3E spectrophotometer (Varian, Inc., Walnut Creek, Calif.) at280 nm to determine the protein concentration. Sample buffer containingβ-mercaptoethanol was added (50 μg of sample per 20 μl of sample buffer)and aliquots were separated on a 10% PAGE/SDS at 80 v for 8 h plus 300v. for 3 h. The gel was silver stained and collagen peptide fragmentswere analyzed by comparison with standard collagen type I fragments. Thecollagen typing of these cultured fibroblasts showed that even after 28days of in vitro culture, the cells retained the same phenotype ascharacterized by collagen type I production.

In vivo implantation of fibroblast-seeded hydrogels. Animal experimentswere carried our according to NIH guidelines for the care and use oflaboratory animals. Male nude mice (n=12) (Simonsen Laboratories Inc.,Gilroy, Calif.), 4-6 weeks old, were reared in the Animal ResourcesCenter at The University of Utah. Under anesthesia, fourfibroblast-seeded HA-DTPH-PEGDA hydrogels were implanted bilaterallyinto subcutaneous pockets surgically prepared in the backs of nude.mice. These served as the experimental group, including 24 implants in 6nude mice, following an approved IACUC protocol. Six additional nudemice received 24 non-cell-loaded HA-DTPH-PEGDA hydrogels as the controlgroup. At each time point (2, 4, and 8 weeks after implantation), fournude mice (two experimentals and two controls) were sacrificed and thespecimens were dissected for macrographical and immunohistochemical(anti-fibronectin) evaluation.

After removal from the mice, the explants appeared more opalescent andelastic with increasing implantation time, suggesting increased celldensity (FIG. 20). The gross examination was confirmed by histology(FIG. 21), by staining for fibronectin production. In controls, after 8weeks of implantation in nude mice, there was no new fibrous tissueformed. In the experimentals, little new fibrous tissue was observed,likely because the cell density for initial seeding was too low, andcell attachment and proliferation factors were added to the implantedgel. Burdick and Anseth (Burdick J A and Anseth K S. Photoencapsulationof osteoblasts in injectable RGD-modified PEG hydrogel for bone tissueengineering. Biomaterials 2002;23:4315-4323) photoencapsulatedosteoblasts in an injectable RGD-modified PEG hydrogel for bone tissueengineering. However the cell number decreased following two weeksculture in vitro. In our case, T31 fibroblast increased tenfold after 28days itt vitro culture, which indicated the injectable hydrogeldescribed here was excellent candidate for tissue regeneration.

V. Preparation of Mitomycin C Hydogel Films

Synthesis of MMC-acrylate. Mitomycin C (2 mg) was dissolved in 10 mldried methylene chloride, and 1.7 μl TEA and 1 μl distilled acryloylchloride were added consecutively (FIG. 22). The reaction mixture wasstirred at room temperature for 4 hours, then concentrated and purifiedby a silica column (methylene chloride:methanol=20:1). The yield is 1.78mg. ¹H NMR (400 MHz, MeOD-d3): δ6.31 (dd, J=2, J=10, 2′-H), 5.82 (dd,J=10,J=2.4, 1H, 3′-H), 5.48 (d, J=0.8, 1H, 3′-H), 4.81 (dd, obscured byMeOH, 1H, 10-H), 4.49 (d, J=13, 1H, 3-H), 3.93 (t, J=11, 1H, 3-H), 3.67(d, J=4.4, 1H, 10-H), 3.64 (d, J=4.8, 1H, 9-H), 3.51 (d, J=12, 1H, 1-H),3.48 (dd, J=1.2, J=4.8, 1H, 2-H), 3.24 (s, 3H, 9a-OCH₃), 1.75 (s, 3H,6-CH₃). ¹³C NMR (400 MHz, MeOD-d3): δ 177.7 (C-1′), 176.1 (C-5), 176.0(C-8), 158.4 (CONH₂), 155.4 (C-4a), 149.7 (C-7), 130.4 (C-2′), 129.4(C-3′), 109.9 (C-8a), 106.0 (C-9a), 103.8 (C-6), 61.5 (C-10), 53.6(C-9), 49.0 (9a-OCH₃), 48.9 (C-3), 42.3 (C-1), 40.9 (C-2), 6.9 (6-CH₃).

Preparation of MMC-HA.

Model Reaction of MMC-Acrylate React with Thiol Group:

The reaction time of MMC-acrylate conjugate to thiol modified HA wasderived by a model reaction. Double protected cysteine was used as amodel reagent to react with MMC-acryloyl. The concentration of thiolgroup was measured using 2-nitro-5-thiosulfobenzoate (NTSB) or Ellmanreagent. The reaction was performed in PBS buffer (pH 8.0) with aconcentration of MMC-acrylate of 0.3 mg/mL and an initial ratio of 2acrylatrs to 1 thiol.Preparation of HA-MMC Conjugate

Thiol-modified HA was prepared using the hydrazide technology describedabove. Briefly, low molecular weight HA (200 k Da) was reacted with3,3′-dithiobis propanoic hydrazide (DTPH) at pH 4.75 by thecarbodiimide-catalysed reaction. The gel like product was reduced bysolid form DTT, after dialysis, the thiolated HA derivatives wereprepared with different loadings. HA-DTPH was dissolved in PBS buffer tothe concentration of 1.25% (w/v). Modified MMC was dissolved in minimalethanol and added into the HA-DTPH solution. The theoretical MMC loadingto the disaccharides was 0.5%, 1% and 2% respectively. The procedure wasconducted under N₂ protection and the final pH of the mixture wasadjusted to 8.0. The reaction was processed for three hours withstirring. FIG. 22 depicts the reaction sequence.

Preparation of HA-MMC-PEG Hydrogel Films

HA-MMC solution was adjusted to pH 7.4 after the coupling reaction. PEGdiacrylate was dissolved in PBS buffer to the concentration of 4.5%(w/v). The two solutions were mixed together and vortexed for oneminute. The reaction mixture was removed by Eppendorf® Combitips andadded to 2 cm×2 cm dishes, 2 mL/dishes. The hydrogels were formed inabout half hour and were evaporated in air to dryness for several daysto form the films. FIG. 23 depicts the reaction sequence.

MMC release experiment. Dried hydrogel films were cut into 2 cm squares.The square gel film and the cut off margin were weighed separately, andthe MMC contained in each square film was calculated. Each film wasdipped into 5 mL 100 mM PBS buffer and shaken gently at 37° C. At eachtime point, 0.5 mL solution was removed and 0.5 mL fresh PBS buffer wasadded. The solution containing released MMC was detected at a wavelengthof 358 nm. The accumulated concentration of released MMC was plotted asa function of the time.

FIGS. 25 a and b show the results of in vitro MMC release results. FIG.25 a shows the absolute released concentration. The released MMC isproportional to the MMC contained in the hydrogel. The relative releasepattern is shown in FIG. 25 b after repoltting the data. HA films with1% and 2% MMC loadings have similar release profiles. At the first halfhour, about 13% MMC was released from the hydrogel, which may come fromtwo sources: one was the uncoupled MMC, the other was hydrolyzed MMC.Then a slow release pattern was observed with a half-life around 48hours. The release of MMC continued for 5 days until reaching aplatform. There were still a considerable amount of MMC embeded in thefilm after 8 days. These results indicate that the newly synthesizedHA-MMC-PEG hydrogel has similar hydrolysis kinetics as the describedMMC-TA conjugate.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

1. A compound having the formula I

wherein Y is a residue of a macromolecule; Q is a SH group or athiol-reactive electrophilic functional group; and L is a polyalkylenegroup, a polyether group, a polyamide group, a polyimino group, an arylgroup, a polyester, or a polythioether group, wherein when Q is a thiolgroup, Y is not a residue of hyaluronan.
 2. The compound of claim 1,wherein the macromolecule comprises an oligonucleotide, a nucleic acidor a metabolically stabilized analogue thereof, a polypeptide, a lipid,a glycoprotein, a glycolipid, or a pharmaceutically-acceptable compound.3. The compound of claim 1, wherein the macromolecule comprises apolysaccharide, a protein, or a synthetic polymer.
 4. The compound ofclaim 1, wherein the macromolecule comprises a polysaccharaide, whereinthe polysaccharide comprises a sulfated-glycosaminoglycan.
 5. Thecompound of claim 4, wherein the polysaccharide comprises chondroitinsulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate,alginic acid, pectin, or carboxymethylcellulose.
 6. The compound ofclaim 1, wherein the macromolecule comprises a synthetic polymer, andthe synthetic polymer comprises glucuronic acid, polyacrylic acid,polyaspratic acid, polytartaric acid, polyglutamic acid, or polyfumaricacid.
 7. The compound of claim 1, wherein the macromolecule comprises aprotein, and the protein comprises a naturally-occurring protein or arecombinant protein.
 8. The compound of claim 1, wherein themacromolecule comprises a protein, and the protein comprises anextracellular matrix protein, a chemically-modified extracellular matrixprotein, or a partially hydrolyzed derivative of an extracellular matrixprotein.
 9. The compound of claim 8, wherein the protein comprisescollagen, elastin, decorin, laminin, or fibronectin.
 10. The compound ofclaim 1, wherein Q is a thiol-reactive electrophilic functional group,wherein the thiol-reactive electrophilic functional group comprises anelectron-deficient vinyl group.
 11. The compound of claim 10, whereinthe electron-deficient vinyl group comprises a nitro group, a cyanogroup, an ester group, an aldehyde group, a keto group, a sulfone group,or an amide group.
 12. The compound of claim 10, wherein thethiol-reactive electrophilic functional group comprises an acrylategroup.
 13. The compound of claim 1, wherein L is CH₂CH₂ or CH₂CH₂CH₂.14. The compound of claim 1, wherein Y is a residue of a protein orpolysaccharide, Q is a thiol, and L is CH₂CH₂ or CH₂CH₂CH₂.
 15. A methodfor coupling two or more thiolated compounds, comprising reacting afirst thiolated compound having the formula III

wherein Y is a residue of a macromolecule, and L is a polyalkylenegroup, a polyether group, a polyamide group, a polyimino group, an arylgroup, a polyester, or a polythioether group, with a second thiolatedcompound having at least one SH group in the presence of an oxidant,wherein the first thiolated compound and second thiolated compound arethe same or different compounds.
 16. The method of claim 15, wherein themacromolecule comprises an oligonucleotide, a nucleic acid or ametabolically stabilized analogue thereof, a polypeptide, a lipid, aglycoprotein, a glycolipid, or a pharnaceutically-acceptable compound.17. The method of claim 15, wherein the macromolecule comprises apolysaccharide, a protein, or a synthetic polymer.
 18. The method ofclaim 15, wherein the macromolecule is a polysaccharide, and thepolysaccharide comprises a sulfated-glycosaminoglycan.
 19. The method ofclaim 18, wherein the polysaccharide comprises chondroitin sulfate,dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginicacid, pectin, or carboxymethylcellulose.
 20. The method of claim 15,wherein Y is a residue of a polysaccharide or a protein and L is CH₂CH₂or CH₂CH₂CH₂.
 21. The method of claim 20, wherein the polysaccharide ishyaluronan.
 22. The method of claim 15, wherein the second thiolatedcompound is a macromolecule comprising an oligonucleotide, a nucleicacid or a metabolically stabilized analogue thereof, a polypeptide, alipid, a glycoprotein, a glycolipid, or a pharmaceutically-acceptablecompound.
 23. The method of claim 15, wherein the second thiolatedcompound comprises a polysaccharide having at least one SH group. 24.The method of claim 15, wherein the second thiolated compound comprisesa sulfated-glycosaminoglycan.
 25. The method of claim 15, wherein thesecond thiolated compound comprises chondroitin sulfate, dermatan,heparan, heparin, dermatan sulfate, heparan sulfate, alginic acid,pectin, carboxymethylcellulose, or hyaluronic acid having at least oneSH group.
 26. The method of claim 15 wherein the second thiolatedcompound comprises a thiolated protein.
 27. The method of claim 15,wherein the second thiolated compound has the formula II

wherein Z is a residue of a macromolecule, and L is a polyalkylenegroup, a polyether group, a polyamide group, a polyimino group, an arylgroup, a polyester, or a polythioether group.
 28. The method of claim27, wherein the macromolecule comprises an oligonucleotide, a nucleicacid or a metabolically stabilized analogue thereof, a polypeptide, alipid, a glycoprotein, a glycolipid, or a pharmaceutically-acceptablecompound.
 29. The method of claim 27, wherein the macromoleculecomprises a polysaccharide, a protein, or a synthetic polymer.
 30. Themethod of claim 27, wherein Z is a residue of hyaluronan and L is CH₂CH₂or CH₂CH₂CH₂.
 31. The method of claim 27, wherein Z is a residue ofgelatin and L is CH₂CH₂ or CH₂CH₂CH₂
 32. The method of claim 15, whereinthe first thiolated compound and the second thiolated compound aredifferent.
 33. The method of claim 15, wherein the oxidant comprises agas comprising oxygen.
 34. The method of claim 33, wherein the oxidantfurther comprises hydrogen peroxide.
 35. A method for making a compound,comprising reacting (a) a first thiolated compound comprising a firstprotein having at least one SH group; and (b) a second thiolatedcompound comprising a polysaccharide or synthetic polymer having atleast one SH group, in the presence of an oxidant.
 36. The method ofclaim 35, wherein the first thiolated compound has the formula III

and the second thiolated compound has the formula II

wherein Y is a protein residue; Z is a polysaccharide residue or aresidue of a synthetic polymer; and each L is, independently, apolyalkylene group, a polyether group, a polyamide group, a polyestergroup, a polyimino group, an aryl group, or a polythioether group. 37.The method of claim 36, wherein L in formula II and II is,independently, CH₂CH₂ or CH₂CH₂CH₂.
 38. The method of claim 36, whereinZ is a residue of hyaluronan.
 39. A compound made by the method of claim15.
 40. A compound having at least one fragment comprising the formulaVI

wherein Y is a residue of a macromolecule; and G is a residue of athiolated compound.
 41. The compound of claim 40, wherein the fragmentcomprises the formula VIII

wherein Y is a residue of a macromolecule, wherein Y is not a residue ofhyaluronan; L is a polyalkylene group, a polyether group, a polyamidegroup, a polyimino group, an aryl group, a polyester, or a polythioethergroup; and G is a residue of a thiolated compound.
 42. The compound ofclaim 41, wherein the macromolecule comprises an oligonucleotide, anucleic acid or a metabolically stabilized analogue thereof, apolypeptide, a lipid, a glycoprotein, a glycolipid, or apharmaceutically-acceptable compound.
 43. The compound of claim 41,wherein the macromolecule comprises a polysaccharide, a protein, or asynthetic polymer.
 44. The compound of claim 41, wherein Y is a residueof a sulfated-glycosaminoglycan.
 45. The compound of claim 41, wherein Yis a residue of chondroitin sulfate, dermatan, heparan, heparin,dermatan sulfate, heparan sulfate, alginic acid, pectin, orcarboxymethylcellulose.
 46. The compound of claim 41, wherein L isCH₂CH₂ or CH₂CH₂CH₂.
 47. The compound of claim 41, wherein G comprises apolysaccharide residue.
 48. The compound of claim 41, wherein Gcomprises a sulfated-glycosaminoglycan residue.
 49. The compound ofclaim 41, wherein G comprises a residue of chondroitin sulfate,dermatan, heparan, heparin, dermatan sulfate, heparan sulfate, alginicacid, pectin, carboxymethylcellulose, or hyaluronan.
 50. A method formaking a compound, comprising reacting a first thiolated macromoleculehaving at least one SH group with at least one compound having at leastone thiol-reactive electrophilic functional group.
 51. The method ofclaim 50, wherein the compound has at least two thiol-reactiveelectrophilic groups.
 52. The method of claim 50, wherein the firstmacromolecule comprises an oligonucleotide, a nucleic acid or ametabolically stabilized analogue thereof, a polypeptide, a lipid, aglycoprotein, a glycolipid, a polysaccharide, a protein, a syntheticpolymer, or a pharmaceutically-acceptable compound.
 53. The method ofclaim 50, wherein the macromolecule comprises a polysaccharide, whereinthe polysaccharide comprises a sulfated-glycosaminoglycan.
 54. Themethod of claim 53, wherein the polysaccharide comprises chondroitinsulfate, dermatan, heparan, heparin, dermatan sulfate, heparan sulfate,alginic acid, pectin, or carboxymethylcellulose.
 55. The method of claim53, wherein the polysaccharide comprises hyaluronan.
 56. The method ofclaim 50, wherein the macromolecule comprises a protein, wherein theprotein comprises an extracellular matrix protein, a partiallyhydrolyzed extracellular matrix protein, or a chemically-modifiedextracellular matrix protein.
 57. The method of claim 56, wherein theprotein comprises collagen, elastin, decorin, laminin, or fibronectin.58. The method of claim 50, wherein the first macromolecule has theformula III

wherein Y is a residue of a macromolecule, and L is a polyalkylenegroup, a polyether group, a polyamide group, a polyimino group, apolyester, an aryl group, or a polythioether group.
 59. The method ofclaim 58, wherein Y is a residue of a polysaccharide or a protein. 60.The method of claim 58, wherein Y is a residue of hyaluronan and L isCH₂CH₂ or CH₂CH₂CH₂.
 61. The method of claim 50, further comprising asecond thiolated macromolecule, wherein the first and secondmacromolecule are the same or different.
 62. The method of claim 50,wherein the thiol-reactive electrophilic functional group comprises anelectron-deficient vinyl group.
 63. The method of claim 62, wherein theelectron-deficient vinyl group comprises a nitro group, a cyano group,an ester group, an aldehyde group, a keto group, a sulfone group, or anamide group.
 64. The method of claim 50, wherein the compound has twoelectron-deficient vinyl groups, wherein the two electron-deficientvinyl groups are the same.
 65. The method of claim 50, wherein thecompound comprises a diacrylate, a dimethacrylate, a diacrylamide, adimethacrylamide, or a combination thereof.
 66. The method of claim 50,wherein the compound has the formula V

wherein R³ and R⁴ are, independently, hydrogen or lower alkyl; U and Vare, independently, O or NR⁵, wherein R⁵ is, independently, hydrogen orlower alkyl; and M is a polyalkylene group, a polyether group, apolyamide group, a polyimino group, a polyester, an aryl group, or apolythioether group.
 67. The method of claim 66, wherein R³ and R⁴ arehydrogen, U and V are oxygen, and M is a polyether group.
 68. The methodof claim 66, wherein R³ and R⁴ are hydrogen, U and V are NH, and M is apolyether group.
 69. The method of claim 66, wherein R³ and R⁴ aremethyl, U and V are oxygen, and M is a polyether group.
 70. The methodof claim 66, wherein R³ and R⁴ are methyl, U and V are NH, and M is apolyether group.
 71. The method of claim 50, wherein the first thiolatedmacromolecule has the formula III

wherein Y is a residue of polysaccharide, and L is CH₂CH₂ or CH₂CH₂CH₂,and the compound has the formula V

wherein R³ and R⁴ are, independently, hydrogen or lower alkyl; U and Vare, independently, O or NR⁵, wherein R⁵ is, independently, hydrogen orlower alkyl; and M is a polyether group.
 72. The method of claim 71,wherein Y is a residue of hyaluronan, and the reaction further comprisesreacting gelatin having at least one thiol group with the compoundhaving the formula V.
 73. The method of claim 71, wherein thepolysaccharide comprises a first polysaccharide and secondpolysaccharide having the formula I, wherein in the firstpolysaccharide, Y is a residue of a first sulfated-glycosaminoglycan,and in the second polysaccharide, Y is a residue of a secondsulfated-glycosaminoglycan, wherein the first and secondsulfated-glycosaminoglycans are the same or different.
 74. The method ofclaim 71, wherein the polysaccharide comprises a first polysaccharideand second polysaccharide having the formula I, wherein in the firstpolysaccharide, Y is a residue of hyaluronan, and in the secondpolysaccharide, Y is a residue of a sulfated-glycosaminoglycan.
 75. Themethod of claim 71, further comprising reacting a protein, anextracellular matrix, or growth factor having at least one thiol groupwith the compound having the formula V.
 76. The method of claim 75,wherein the polysaccharide comprises a sulfated-glycosaminoglycan. 77.The method of claim 50, wherein the first thiolated macromolecule hasthe formula III

wherein Y is a residue of polysaccharide, and L is CH₂CH₂ or CH₂CH₂CH₂,and the compound is mitomycin C modified with an acrylate group.
 78. Amethod for making a compound, comprising reacting a thiolatedmacromolecule having at least one thiol-reactive electrophilicfunctional group with at least one compound having at least two thiolgroups.
 79. The method of claim 78, wherein the first thiolatedmacromolecule has the formula I

wherein Y is a residue of the macromolecule; Q is the thiol-reactiveelectrophilic functional group; and L is a polyalkylene group, apolyether group, a polyamide group, a polyimino group, a polyester, anaryl group, or a polythioether group.
 80. The method of claim 79,wherein Y is a residue of a polysaccharide.
 81. The method of claim 79,wherein Y is hyaluronan and L is CH₂CH₂ or CH₂CH₂CH₂.
 82. The compoundproduced by the process of claim
 50. 83. A compound having at least onefragment comprising the formula VII

wherein R⁷ and R⁸ are, independently, hydrogen or lower alkyl; X is anelectron-withdrawing group; and Y is a residue of a macromolecule. 84.The compound of claim 83, wherein Y is a polysaccharide residue.
 85. Thecompound of claim 83, wherein the fragment comprises the formula IV

wherein R³ and R⁴ are, independently, hydrogen or lower alkyl; U and Vare, independently, O or NR⁵, wherein R⁵ is, independently, hydrogen orlower alkyl; Y is a residue of a protein; Z is a polysaccharide residueor a residue of synthetic polymer; and M is a polyalkylene group, apolyether group, a polyamide group, a polyester group, a polyiminogroup, an aryl group, or a polythioether group.
 86. The compound ofclaim 85, wherein Y has the formula IX

wherein Y′ is a residue of the first protein; L is a polyalkylene group,a polyether group, a polyamide group, a polyester group, a polyiminogroup, an aryl group, or a polythioether group, wherein the L group iscovalently bonded to the sulfur atom.
 87. The compound of claim 86,wherein the protein comprises an extracellular matrix protein, apartially hydrolyzed extracellular matrix protein, or achemically-modified extracellular matrix protein.
 88. The compound ofclaim 86, wherein the protein comprises collagen, elastin, decorin,laminin, or fibronectin.
 89. The compound of claim 85, wherein Z has theformula X

wherein Z′ is a polysaccharide residue or a residue of a syntheticpolymer; and L is a polyalkylene group, a polyether group, a polyamidegroup, a polyester group, a polyimino group, an aryl group, or apolythioether group, wherein the L group is covalently bonded to thesulfur atom.
 90. The compound of claim 89, wherein Z is a residue ofhyaluronan or chondroitin sulfate and L is CH₂CH₂ or CH₂CH₂CH₂.
 91. Apharmaceutical composition comprising a pharmaceutically-acceptablecompound and the compound of claim
 40. 92. A pharmaceutical compositioncomprising a living cell and the compound of claim
 40. 93. A method forimproving wound healing in a subject in need of such improvement,comprising contacting the wound of the subject with the compound ofclaim
 40. 94. A method for delivering at least onepharmaceutically-acceptable compound to a patient in need of suchdelivery, comprising contacting at least one tissue capable of receivingthe pharmaceutically-acceptable compound with the composition of claim91.
 95. A method for delivering living cells to a patient in need ofsuch delivery, comprising contacting at least one tissue capable ofreceiving the living cells with the composition of claim
 92. 96. The useof the compound of claim 40 as a growth factor, an anti-inflammatoryagent, an anti-cancer agent, an analgesic, an anti-infection agent, oran anti-cell attachment agent.
 97. A compound having the formula XI

wherein J comprises a protein residue; and E comprises a fluorescenttag, a radiolabel, a targeting moiety, a lipid, a peptide, aradionuclide chelator with a radionuclide, a spin-label, a PEGcamouflage, a metal surface, a glass surface, a plastic surface, or acombination thereof.
 98. The compound of claim 97, wherein themacromolecule comprises a protein, and the protein comprises anaturally-occurring protein or a recombinant protein.
 99. The compoundof claim 97, wherein the protein comprises an extracellular matrixprotein, a partially hydrolyzed extracellular matrix protein, or achemically-modified extracellular matrix protein.
 100. The compound ofclaim 97, wherein the protein comprises collagen, elastin, decorin,laminin, or fibronectin.
 101. A compound produced by the processcomprising reacting (1) a protein having at least one hydrazide-reactivegroup and (2) a compound having at least one hydrazide group.
 102. Acompound produced by the process comprising reacting (1) a proteinhaving at least one hydrazide group and (2) a compound having at leastone hydrazide-reactive group.
 103. A pharmaceutical compositioncomprising a pharmaceutically-acceptable compound and the compound ofclaim
 97. 104. A method for improving wound healing in a subject in needof such improvement, comprising contacting the wound of the subject withthe compound of claim
 97. 105. A method for delivering at least onepharmaceutically-acceptable compound to a patient in need of suchdelivery, comprising contacting at least one tissue capable of receivingthe pharmaceutically-acceptable compound with the composition of claim104.
 106. The use of the compound of claim 97 as a growth factor, ananti-inflammatory agent, an anti-cancer agent, an analgesic, ananti-infection agent, or an anti-cell attachment agent.
 107. A kitcomprising (1) a compound comprising at least one hydrazide group; (2) acondensing agent; (3) a buffer reagent; and (4) a purification column.108. A pharmaceutical composition comprising apharmaceutically-acceptable compound and the compound of claim
 82. 109.A pharmaceutical composition comprising a pharmaceutically-acceptablecompound and the compound of claim
 83. 110. A pharmaceutical compositioncomprising a living cell and the compound of claim
 82. 111. Apharmaceutical composition comprising a living cell and the compound ofclaim
 83. 112. A method for improving wound healing in a subject in needof such improvement, comprising contacting the wound of the subject withthe compound of claim
 82. 113. A method for improving wound healing in asubject in need of such improvement, comprising contacting the wound ofthe subject with the compound of claim
 83. 114. The use of the compoundof claim 82 as a growth factor, an anti-inflammatory agent, ananti-cancer agent, an analgesic, an anti-infection agent, or ananti-cell attachment agent.
 115. The use of the compound of claim 83 asa growth factor, an anti-inflammatory agent, an anti-cancer agent, ananalgesic, an anti-infection agent, or an anti-cell attachment agent.